Chapter Two

2. Plant tissue culture

2.1. Introduction
Plants are key to life on earth
 Supply 90% of human calorie intake
 80% of the protein intake
 There are about 3000 plant species used by man as food
 The world now depends mainly on about 20 crop species for the majority of its
calories
 50% of calories is contributed by eight species of cereals
 Minerals and vitamins are supplied by a further 30 species of fruits and
vegetables
 More than one-third of all cultivated land is used to cultivate wheat and rice
 Cont.…
 It has been calculated that the earth can support about 15 billion people
on a strictly vegetarian diet
The farming practices and crops cultivated today have developed over a
relatively short span of time
 Crop plants of today have changed in a number of ways
 Therefore, they now bear very little resemblance to their wild type
ancestors
 Today, varieties are the result of generation of plants cultivated under
ideal conditions from the man’s point of view
 From the beginning of crop cultivation to the late 19 th cent. all the

improvements in the species used were brought about by farmers

 Since then,

1. The law of genetic inheritance laid down by Mendel and

2. Rules governing species variation by Darwin and others redefined the

breeding techniques
 These techniques made them

- predictable and quicker

- more precise and more productive

 Despite the implementation of breeding technique, the time taken to

produce and test varieties is an important limiting consideration
 New Technologies

 Over the past few years, a number of methodologies have been developed in
advancing research in plant science
 These could be used to develop new crops
1. Manipulation and subsequent growth of cells, tissues, organs and protoplasts
in tissue culture
2. Genetic engineering (recombinant DNA technology)

 These two areas of research have in recent years become associated with the
general field of Biotechnology
 Plant Tissue Culture
 The term plant tissue culture broadly refers to the in vitro cultivation of

plants, seeds, plant parts on nutrient media under aseptic conditions

 It is based on the cell doctrine that states a cell is capable of autonomy

and is potentially totipotent
 History

 In 1902 – Gottlieb Haberlandt (1854-1945) attempted to cultivate plant cells

in vitro
 He used single cells isolated from:
 palisade tissue of Lamium purpureum
 pith cells from petioles of Eicchornia crassipes
 grandular hair of Pulminaria and Utrica
 stamen hair cells of Tradescantia
 Stomatal guard cells of Ornithogalum, and
 the other plant materials
 He grew them on Knop’s salt solution with glucose, sucrose and peptone
 observed obvious growth in the palisade cells
 He developed the concept of in vitro cell culture

 He was the first to culture isolated, fully differentiated cells in a nutrient

medium
 Realized that asepsis was necessary when culture media are enriched with
organic substances metabolized by microorganisms
 In his cultures, cells were able to synthesize starch as well as increase in size
and survived for several weeks
 However, he failed in his goal to induce these cells to divide
 Despite drawbacks, he made several predictions about the requirements
for cell division under experimental conditions in 1902
 This have been confirmed with the passage of time
 Haberlandt is thus regarded as the father of tissue culture
• Skoog (1944), Skoog and Tsui (1951) demonstrated that adenine stimulates

cell division and induces bud formation in tobacco tissue even in the
presence of IAA
• This convinced Skoog and collaborators those nucleic acids which contain
substances such as adenine influence tissue proliferation
• In 1955, Skoog and collaborators finally isolated a derivative of adenine (6-
furfyl aminopurine), named kinetin from autoclaved yeast extract
• A substance with kinetin-like properties was also detected in young maize
endosperm which was isolated by Letham (1963) and named zeatin
• It was also verified that a similar substance called ribosylzeatin found in
coconut milk (Letham, 1974)
• Now many synthetic as well as natural compounds with kinetin-like activity
are known which show bud-promoting properties
• These substances are collectively called cytokinins
• They are used to induce divisions in cells of highly mature and differentiated
tissues
 Laboratory Organization and Facilities

 A suitable culture medium and proper culture conditions must be provided

for a plant to express its intrinsic or induced potential

 The overall design in any laboratory organization must focus on maintaining

aseptic conditions

 The specific design can vary

 But an effective laboratory organization for plant tissue culture must include
certain elements irrespective of whether used by a scientist or group of
workers
 The size and proportion of different parts of the facility will be dependent
upon the

- function

- purpose

- size of the operations

 The basic rule essential in the planning of an efficient and functional

laboratory facility is:

 The order and continuous maintenance of cleanliness
 Because all operations require aseptic conditions
 A general guideline for setting up a facility is:
 To focus on a design that different units and activities need to be
arranged to make operational steps possible with the least amount of
cross-traffic

 Thus a tissue culture facility should have the following features

 Laboratory and personal safety

 Every new worker should be given some orientation before starting work

 It should include instructions on

 How to operate various equipments

 Special instructions for fire, broken glass, chemical spills or accidents

with sharp-edged instruments
 Tissue culture facilities

Washing facility

 An area with large sinks and draining areas

 The conventional method for cleaning laboratory glassware involves
- chromic acid-sulphuric acid soak followed by thorough washing with
tap water and subsequent rinsing with distilled water
 However, due to the corrosive nature of acids, it is recommended that for
routine procedures glassware should be
 soaked in a 2% detergent cleaner for 16 h followed by
 washing with 60-70C hot tap water, and finally distilled water
 Some precautions should be taken during acid cleaning
i. Handling of acid and processing of glassware in the acid should be
done in a fume hood
ii. Never add water to acid but for dilution acid should be added
slowly to the water while stirring
iii. After acid cleaning, if the solution in the acid becomes dark colored,
it should be discarded
- However, with the availability of a wide range of reusable
plasticware, it is recommended that these should be washed
with mild detergents followed by a rinse with tap water and
then distilled water
General laboratory and media preparation area

 This is the area where most of the activities are performed

 It includes area for
 media preparation
 autoclaving media
 many of the activities related to the handling of tissue culture materials
 should have ample storage and bench space for chemicals, glassware,
culture vessels, closures and other items needed to prepare media
 Transfer area

 A sterile dust-free room should be available for routine transfer and

manipulation work
 The laminar airflow cabinet is the most common accessory used for aseptic
manipulations
 It is cheaper and easier to install than a transfer room
 Should be equipped with gas cocks if gas burners are to be used or with glass
bead sterilizers
 In airflow cabinet, air is forced into a cabinet through a bacterial HEPA filter
 It flows outwards (forward) over the working bench at a uniform rate
 The roof of some of the cabinets contain UV germicidal light often used to
sterilize the interior chamber
 Culturing facilities

 Plant tissue cultures should be incubated under conditions of well-controlled

 temperature
 illumination
 photoperiod
 humidity and air circulation

 Incubation culture rooms

 Commercially available cabinets
 Large plant growth chambers
 Walk-in environmental rooms satisfy this requirement
 These facilities can also be constructed by developing a room with proper

 air conditioning

 perforated shelves

 fluorescent tubes

 a timing device to set photoperiods

 a dark area closed off from the rest of room with thick black curtains is
necessary if some cultures require continuous darkness
Greenhouses

 In the greenhouse, plants are acclimatized before being transferred to the

field
 The plants are grown in the greenhouse to develop adequate root systems
and leaf structures to withstand field environment
 The greenhouse should be equipped with
 cooling and heating systems
 artificial lighting system
 misting system
 Sterilization techniques

 Sterilization is a procedure used for elimination of microorganisms

 The need for asepsis requires that all culture vessels, media, and
instruments used in handling tissues, as well as explant itself be sterilized

 All operations are carried out in laminar airflow cabinets

 The precaution is not to share these cabinets of tissue culture work with
other microbiologists and pathologists
 In general, different sterilization procedures can be grouped under three
categories

1. Preparation of sterile media, containers and small instruments

2. Maintenance of aseptic condition

3. Preparation of sterilized explant material

 Preparation of sterile media, containers and small instruments

 The most popular method of sterilizing culture media and apparatus is by

autoclaving the material using steam/dry sterilization

Steam sterilization

 Most nutrient media are sterilized by using an autoclave

 The standard conditions for autoclaving media are

 121 C with a pressure of 105 kPa for 15 min

 Good sterilization relies on time, pressure, temperature and volume of

the object to be sterilized
 During autoclaving, bottles should not be tightly packed and their tops

should be loose

 After autoclaving, these are kept in laminar airflow cabinet and tops of
bottles are tightened when they are cool

 The advantages of an autoclave are speed, simplicity and destruction of

viruses

 The disadvantages are change in pH by 0.3-0.5 units, components can

separate out and chemical reactions can occur resulting in a loss of activity of
media constitutes
During and after autoclaving, the following points should be considered
i. The pH of the media is lowered by 0.3-0.5 units
ii. Autoclaving at too high temperature can caramelize sugars, which may be
toxic
iii. Volatile substances can be destroyed by the use of an autoclave
iv. Autoclaving for too long periods can precipitate salts, and depolymerize the
agar
v. Care should be taken to use the correct duration and temperature
vi. For sterilization of liquid material, wet or steam sterilization employing
either autoclave or domestic pressure cooker can be used
Dry sterilization
 The sterilization of glassware and metallic instruments can be carried out in
dry heat for 3 h at 160-180 C
 Dry goods can either be wrapped in aluminum foil, brown paper or sealed
metal containers to maintain sterility
 Present day autoclaves come with both dry and steam sterilization programs
 Maintenance of aseptic conditions

Procedures/techniques employed during aseptic transfer

Alcohol sterilization

• Wash with antibacterial detergent – spraying with 70% alcohol on hands

• The laminar airflow cabinets should also be sprayed with 70% ethanol before
use

Flame sterilization

• Used for sterilization of instruments that are continuously used during

manipulation work
• Instruments are soaked in 70% alcohol followed by flaming on a burner in the
airflow hood or
• Heat sterilization in glass beads
• Culture containers should be covered as quickly as possible after an operational
step is completed
• Talking in the hood should be avoided
• Set up all materials including containers, media, etc. in one side of the cabinet
• It should not disturb the airflow pattern
 Sterilization of Explant

Chemical sterilization
• Plant material can be surface sterilized by a variety of chemicals
• It is the eradication of microorganisms with the aid of chemicals
• The type and concentration of chemical sterilant to be used and exposure
time must be decided upon empirically
Some commonly used chemical sterilants
• Sodium hypochlorite
• Calcium hypochlorite
• Hydrogen peroxide
• Bromine water
• Silver nitrate
• Mercuric chloride
• Antibiotics
1. 1% solution of sodium hypochlorite - (NaClO), commercial bleach:
- It is generally available with 5% active chlorine content, so 20% can be
used for normal sterilization
2. Calcium hypochlorite – Ca(ClO)2:
- This comes in powder form
- It is mixed well with water, allowed to settle and the clarified filtered
supernatant solution is used for sterilization
- Generally, 4-10g/100 ml of Ca (ClO)2 is used
- It enters the plant tissue slowly as compared to sodium hypochlorite
- It can be stored only for a limited period of time because of
deliquescent (takes up water) nature
3. 1% solution of bromine water
4. 0.01 to 1% solution of mercuric chloride:
- It is dissolved in water to make solution
- It is an extremely toxic substance for plants, so rinsing must be
very thorough
- It should be disposed off in a separately marked container
5. Alcohol:
- 70% alcohol is used for sterilization of plant material by dipping
them for 30 sec to 2 min
- Generally, alcohol alone is not sufficient to kill all the
microorganisms and the plant material after alcohol treatment is
treated with another chemical sterilant
6. 10% hydrogen peroxide
7. 1% silver nitrate solution
8. 4-50 mg/l of antibiotic solution
 • It is important that the plant surface be properly wetted by the sterilizing

solution
• A 30 sec immersion in 70% (v/v) ethanol or the addition of a few drops of
liquid detergent (e.g. Teepol, Tween 20) to the other chemical sterilant
solutions during use will ensure effectiveness of the sterilizing agent

 After treatment with sterilants, explants must be thoroughly rinsed with

several changes of sterile distilled water

 Tissue culture medium
 Nutritional requirement for optimal growth of a tissue
invitro may vary with the species.

 Even tissue from different parts of a plant may have

different requirements for satisfactory growth.

 As such, no single medium can be suggested as being

entirely satisfactory for all types of plant tissues and organs.

 When starting with a new system, it is essential to work out

a medium that will fulfill the specific requirements of that
tissue.
 Types of medium
Heller’s Medium
MS (Murashige and Skoog) Medium
ER (Eriksson) Medium
B5 Medium
Nitsch’s Medium
NT Medium
White Medium
• A nutrient medium is defined by its
• mineral salt composition
• vitamins
• carbon source
• phytohormones and
• other organic supplements
 Media composition

 There are several basic media

 The MS medium of Murashige and Skoog (1962) salt composition is very

widely used in different culture systems

 In the development of this medium and also other media, it was

demonstrated that

- the presence of necessary nutrients and

- the actual and relative concentrations of various inorganic nutrients are of

crucial significance
Inorganic nutrients
 Mineral elements are very important in the life of a plant
 Ca – a component of cell wall
 N – an important part of amino acids, nucleic acids and vitamins
 Mg – part of chlorophyll molecules
 Fe
 Zn
 Mb
are parts of certain enzymes
 C, H, N, O, 12 other elements – essential for plant growth
 According to the recommendations of IAPP, the elements required by plants in

concentration greater than 0.5 mmol/l are referred to as macroelements

 Those required in concentration less than that are microelements

 A variety of salts supply the needed macro/major and micro/minor nutrients

that are the same as those required by the normal plant
Carbon and energy source

 The standard carbon source is sucrose

 But plant tissues can utilize a variety of carbohydrates such as glucose,

fructose, lactose, maltose, galactose and starch

 In the cultured tissues or cells, photosynthesis is inhibited and thus

carbohydrates are needed for tissue growth in the medium

 The sucrose in the medium is rapidly converted into glucose and fructose

 Glucose is then utilized first, followed by fructose

 Sucrose is generally used at 2-5%

 Most media contain myo-inositol at a concentration of about 100 mg/l, which

improves cell growth
Vitamins

 Plant cells in culture have a requirement for vitamins

 There is an absolute requirement for vitamin B1 (thiamine), nicotinic acid

and vitamin B6 (pyridoxine)

 Some media contain pantothenic acid, biotin, folic acid, p-amino benzoic
acid, choline chloride, riboflavin and ascorbic acid

 The concentrations are in the order of one mg/l

 Myo-inositol is another vitamin used in the nutrient medium with a

concentration of the order 10-100 mg/l
Growth regulators

 Hormones are organic compounds naturally synthesized in higher plants

 They are usually active at a site different from where they are produced

 Present and active in very small quantities

 Synthetic compounds that correspond to the natural ones have also been
developed

 Two main classes of growth regulators that are special importance in TC

 Others are of minor importance

 Auxins

 Common feature is their property to induce cell division and formation of callus

 Causes – cell division

- cell elongation

- swelling of tissues

- formation of callus

- the formation of adventitious roots

- it inhibits adventitious and axillary shoot formation

 At low concentrations, adventitious root formation predominates

 At high concentrations, callus formation takes place
• Auxin is present in sufficient concentration in the growing shoot tips or

flowering tips of plants to ensure multiplication and elongation

• Auxin circulates from the top towards the base of the organs with a polarity
strongly marked in young organs

These auxins are

 2, 4-D
 NAA
 IAA
 IBA
 2, 4, 5-T
 pCPA
 picloram
Cytokinins
 Derivatives of adenine
 Have an important role in shoot induction
 They promote – cell division if added together with auxins
- at higher concentrations, induce adventitious shoot
- inhibits root formation

 Promote axillary shoot formation by decreasing apical dominance

 Most frequently used cytokinins are
 kinetin
 BAP
 BA
 Zeatin
 2-ip (isopentenyladenine)
Other growth regulators

 Gibberellins, although found in all plants and fungi, are unevenly

distributed

 The sites of synthesis are very young , unopened leaves, active buds, root
tips and embryos

 Gibberellins are normally used in plant regeneration

 GA3 is essential for mersitem culture of some species

 In general, gibberellins induce elongation of internodes and the growth of
meristems or buds in vitro

 Gibberellins usually inhibit adventitious root as well as shoot formation

 During organogenesis, gibberellins are antagonistic

 They seem to oppose the phenomenon of dedifferentiation

• Abscisic acid is an important growth regulator for induction of embryogenesis
• This is a growth inhibitor, which seems to be synthesized when a plant is
under difficult conditions
• It has a favorable effect on abscission
• Ethylene is a gaseous compound indentified a long time ago
• But its functions as a growth regulator were not evident
• Ethylene is produced by cultured cells
• But its role in cell and organ culture is not known
• The practical use of ethylene, which is difficult in a gaseous state, made great
progress after the discovery of 2-chloroethane phosphoric acid
• This product, when applied in a powder form, penetrates the tissue where it
liberates ethylene
Anti browning compounds
• Many plants are rich in polyphenolic compounds
• After tissue injury during dissection, such compounds will be oxidized by
polyphenol oxidases and the tissue will turn brown or black
• The oxidation products are known to inhibit enzyme activity, kill the explants,
and darken the tissues and culture media, a process which severely affects the
establishment of explants
• Activated charcoal is generally acid washed and neutralized at concentrations
of 0.2 to 3.0% (w/v) in the medium where phenol like compounds are a
problem for in vitro growth of cultures
Gelling agents

 Agar, a seaweed derivative, is the most popular solidifying agent

 It is a polysaccharide with high MW and has the capability of gelling media
 Solubilized agar forms a gel that can bind water and adsorb compounds
 The higher the agar concentration, the stronger is the binding of water
 Plant tissue culturists often use Difco Bacto agar at a concentration of 0.6 to
1.0% (w/v)
 Other forms of agar (agarose, phytagar, flow agar, etc.) are also becoming
popular
 In vitro growth may be adversely affected if the agar concentration is too
high
 With higher concentrations, the medium becomes hard and does not allow
the diffusion of nutrients into the tissues
 Impurities, if present in the agar, can be removed for critical experiments on
nutrient studies
pH

• pH determines many important aspects of the structure and activity of

biological macromolecules

• Nutrient medium pH ranges from 5 to 6

• pH higher than 7.0 and lower than 4.5 generally stops growth and
development

• The pH before and after autoclaving is different

• It generally falls by 0.3 to 0.5 units after autoclaving

• If the pH falls appreciably during plant tissue culture (the medium becomes

liquid), then a fresh medium should be prepared

• It should be known that a starting pH of 6.0 could often fall to 5.5 or even
lower during growth

• pH higher than 6.0 gives a fairly hard medium and a pH below 5.0 does not
allow satisfactory gelling of the agar
 Quiz
1. How do you define the term sterilization?
2. What is the disadvantage using autoclave as
sterilization equipment?
3. What is the common carbon source for in vitro
culture?
4. List two most important growth regulators used in
tissue culture?
5. what is a chemical used to solidifying the media?
 Types of Culture

 The procedures of plant tissue culture have developed to such a level that any
plant species can be regenerated in vitro through several methodologies

 Unlike animal cells, plant cells retain the ability to change to a meristematic
state and

 Differentiate into a whole plant if it has retained an intact membrane system

and viable nucleus
 In PTC more often we use an explant to initiate their growth in culture

 The non-dividing, differentiated, quiescent cells of the explant first undergo

changes to achieve meristematic state when grown on a nutrient medium
 The phenomenon of mature cells reverting to a meristematic state and

forming undifferentiated callus tissue is termed as dedifferentiation

 Since the multicellular explant comprises cells of diverse types, the callus
derived will be heterogeneous

 The ability of the component cells of the callus to differentiate into a whole
plant or a plant organ is termed as redifferentiation
 Types of Culture

Embryo culture Seed culture Meristem culture

Plant Tissue Culture

Cell culture Protoplast culture

Organ culture
Callus culture Bud culture
 Callus culture
 Non-organized tumor tissue
 Arises on wounds of differentiated tissues and organs
 When critically examined, callus culture is not homogeneous mass of cells,
 Because it is usually made up of two types of tissues
 Differentiated and
 Non-differentiated
 Roots, stems, leaves, flowers, etc. are used as explant for callus induction
 If there are only differentiated cells present in an isolated explant, then
dedifferentiation must take place
 Parenchyma cells present in the explant usually undergo this differentiation
 If the explant already contains meristematic tissue when isolated,
 Then this can divide immediately without dedifferentiation taking place
 Dedifferentiation plays a very important role
 Enables mature cells in an explant isolated from an adult plant to be
redetermined
 In this process, adult cells are temporarily able to revert from the adult to
the juvenile state

 The rejuvenated cells have a greater growth and division potential and

 Under special circumstances are able to regenerate into organs and/or

embryos

 Is formed under the influence of exogenously supplied GRs

 Many other factors are important for callus formation:
 Genotype
 Composition of the nutrient medium
 Physical growth factors (light, temperature, etc.)

 The effect of light on callus formation is dependent on the plant species

 Light may be required in some cases and darkness in other cases

 Anther culture

• This technique is simple, quick and efficient

• Young flower with immature anthers at appropriate stage of

development are surface sterilized and rinsed with sterile water
• The calyx from flower buds is removed by forceps
• The corolla is slit open and stamens are removed and placed in a
sterile Petri dish
• Each anther is gently separated from the filament and
• The intact uninjured anthers are cultured horizontally on nutrient
media
• Discard injured anthers because wounding often stimulates
callusing of the anther wall tissue
• When working with plants having minute flowers such as Brassica
and Trifolium, it may be necessary to use a stereo microscope for
dissecting the anthers
• Anthers can be plated on solid agar media in Petri dishes
 Significance and uses of haploids

1. Dev’t of pure homozygous lines

2. Hybrid dev’t
3. Induction of mutations
4. Induction of genetic variability
5. Generation of exclusively male plants
6. Cytogenetic research
7. Significance in the early release of varieties
8. Hybrid sorting in haploid breeding
9. Disease resistance
10. Insect resistance
11. Salt tolerance
12. Doubled haploids in genome mapping
 Protoplast culture
 Protoplasts contain all the components of a plant cell except for the cell
wall.

 Using protoplasts, it is possible to regenerate whole plants from single

cells and also develop somatic hybrids.

 Cell walls can be removed from explant tissue mechanically or

enzymatically; the latter is used most often.

 Plant cell walls consist of cellulose, hemicellulose, and pectin, with

lesser amounts of protein and lipid.

 Hence a mixture of enzymes is necessary for degrading the cell wall.

The enzymes that are commonly used are cellulase and pectinase.
 Protoplast culture

• Protoplasts are isolated and cultured

• Are fused with other species to form hybrid cells

• Somatic hybridization – has application to produce hybrid plants
 Embryo culture

 The sterile isolation and growth of an immature or mature embryo

Mature embryo culture

 The culture of mature embryos derived from ripe seeds

 This type of culture is done when:

 embryos do not survive in vivo
 become dormant for long periods of time
 to eliminate the inhibition of seed germination
• Seed dormancy of many species is due to chemical inhibitors or mechanical

resistance present in the structures covering the embryo, rather than

dormancy of the embryonic tissue
• Excision of embryos from the testa and culturing them in the nutrient media
may bypass such seed dormancy
• Some species produce sterile seeds, which may be due to incomplete embryo
development
• Embryo culture procedures may yield viable seedlings
• Embryos excised from the developing seed at or near the mature stage are
autotrophic and grown on a simple inorganic medium with a supplemental
energy source
Immature embryo culture (embryo rescue)

 The culture of immature embryos to rescue embryos of wide crosses

 Mainly used to avoid embryo abortion

 Since there are various barriers at pre-and post-fertilization levels

 The pre-fertilization barriers include all factors that hinder effective

fertilization, which is usually due to inhibition of pollen tube growth by the
stigma or upper style
 Post-fertilization barriers hinder or retard the development of the zygote after

fertilization and normal development of the seed

• This frequently results from the failure of the hybrid endosperm to develop
properly
• Leading to starvation and abortion of the hybrid embryo or
• Results from embryo-endosperm incompatibility where the endosperm
produces toxins that kills the embryo
• Endosperm development precedes and support embryo development
nutritionally, and
• Endosperm failure has been implicated in numerous cases of embryo abortion
• The underlying principle of embryo rescue techniques is the aseptic isolation

of embryo and its transfer to a suitable medium for development under

optimum culture conditions
• With embryo culture, there are normally no problems with disinfection
• Florets are removed at the proper time and either florets or ovaries are
sterilized
• Ovules can then be removed from the ovaries
• The tissue within the ovule, in which the embryo is embedded, is already
sterile
• For mature embryo culture either single mature seeds are disinfected or
• If the seeds are still unripe, then the still closed fruit is disinfected
• The embryos can then be aseptically removed from the ovules
• Utilization of embryo culture to overcome seed dormancy requires a different
procedure
• Seeds that have hard coats are sterilized and soaked in water for few hours to
few days
• Sterile seeds are then split and the embryos excised
 Applications of embryo culture

1. Prevention of embryo abortion in wide crosses

2. Production of haploids
3. Overcoming seed dormancy (e.g. Musa balbisiana)
4. Shortening of breeding cycle
5. Prevention of embryo abortion with early ripening stone fruits
6. In vitro clonal propagation
7. Germination of seeds of obligatory parasites without host is impossible
in vivo, but is achievable with embryo culture
 Meristem Culture
 In addition to being used as a tool for plant propagation, tissue
culture is a tool for the production of pathogen-free plants.

 Using apical meristem tips, it is possible to produce disease-

free plants.

 This technique is referred to as meristem culture, meristem tip

culture, or shoot tip culture, depending on the actual explant
that is used.

 Although it is possible to produce bacterium- or fungus-free

plants, this method has more commonly been used in the
elimination of viruses in many species.
 Cont..
 Apical meristems in plants are suitable explants for the
production of virus-free plants

 since the infected plant’s meristems typically harbor titers that

are either nearly or totally virus-free.

 Meristem culture in combination with thermotherapy has

resulted in successful production of virus-free plants when
meristem culture alone is not successful
 REGENERATION METHODS OF PLANTS IN CULTURE
 In plant biotechnology, tissue culture is most important for the
regeneration of transgenic plants from single transformed
cells.

 It is safe to say that without tissue culture there would be no

transgenic plants.
1. Organogenesis
2. Somatic Embryogenesis
 Organogenesis
• Organogenesis is the formation of organs: either shoot or root.

• Organogenesis in vitro depends on the balance of auxin and

cytokinin and the ability of the tissue to respond to
phytohormones during culture.

• Organogenesis takes place in three phases. In the first phase

the cells become competent; next, they dedifferentiate.

• In the third phase, morphogenesis proceeds independently of

the exogenous phytohormone.
• Organogenesis in vitro can be of two types: direct and indirect
 Cont..
1. Indirect Organogenesis. Formation of organs indirectly via a
callus phase is termed indirect organogenesis.

• Induction of plants using this technique does not ensure clonal

fidelity,

• but it could be an ideal system for selecting somaclonal

variants of desired characters and also for mass multiplication.

2. Direct Organogenesis. The production of direct buds or shoots

from a tissue with no intervening callus stage is termed direct
organogenesis
 Somatic Embryogenesis
 Somatic embryogenesisis a nonsexual developmental process
that produces a bipolar embryo with a closed vascular system
from somatic tissues of a plant.

 Somatic embryogenesis has become one of the most powerful

techniques in plant tissue culture for mass clonal propagation.
 Micropropagation

Seed vs soma

Propagation using seeds

 Seeds have several advantages as a means of propagation
 They are often produced in large numbers
 They may be stored for long periods without loss of viability
 Easily distributed

 Plant grown from seed are without most of the pest and diseases which may
have afflicted their parents

 However, for many agricultural and horticultural purposes it is desirable to

cultivate clones or populations of plant which are practically identical
 However, the seeds of many plants typically produce plant which differ
genetically, and

 To obtain seeds which will give uniform offspring is either very difficult or
impossible in practical terms

 Genetically uniform populations of plant can result from seeds in three

ways:

1. From inbred (homozygous) lines

2. From F1 seeds (produced by crossing two homozygous parents)

3. From apomictic seedlings

 Vegetative propagation

 Suitable methods for vegetative propagation have been developed over many

cent.

 These traditional ‘macro-propagation’ techniques utilize relatively large pieces

of plants

 These methods have been refined and improved by modern horticultural

research
 In vitro propagation

Advantages

 Cultures are started with very small pieces of plants (explants)

 Only a small amount of space is required to maintain plants or to increase their

number

 Propagation is ideally carried out in aseptic conditions, free from pathogens

 To produced virus-free plants

 A more flexible adjustment of factors influencing vegetative regeneration is
possible

 It may be possible to produce clones of some kinds of plants that are

otherwise slow and difficult to propagate vegetatively

 Plants may acquire a new temporary characteristic through micropropagation

that makes them more desirable

 Production can be continued all the year round and is independent of

seasonal changes
Disadvantages

 The chief disadvantages of in vitro methods are that

 advanced skills are required

 a specialized and expensive production facility is needed

 Fairly specific methods may be necessary to obtain optimum results from

each species and variety

 Therefore, the cost of propagules is usually high

 The plantlets obtained are initially small and sometimes have undesirable

characteristics

 In order to survive in vitro, explants and cultures have to be grown on a

medium containing sucrose or some other carbon source

 Young plantlets are more susceptible to water loss in an external environment

 They may therefore have to be hardened in an atmosphere of slowly

decreasing humidity and increased light

 The chances of producing genetically aberrant plants may be increased

 Stages of micropropagation

 Murashige defined three stages (I-III) in the in vitro multiplication of plants

 Some workers have suggested other stages (Stage 0 and stage IV)

Stage 0: Mother plant selection and preparation

 Before micropropagation commences, careful attention should be given to the

selection of a stock plant or plants

 They must be typical of the variety or species, and free from any symptoms of
disease
 Stage II: The production of suitable propagules

 The stage to bring about the production of new plant outgrowths or

propagules
 Multiplication can be brought about from
 Newly-derived axillary or adventitious shoots
 Somatic embryos
 Miniature storage or propagative organs
Stage III: Preparation for growth in the natural env’t

 Steps are taken to grow individual plantlets capable of carrying out

photosynthesis, and

 Survival without an artificial supply of carbohydrate

 This stage is often divided into

Stage IIIa

- Elongation

Stage IIIb

- Rooting
 Stage IV: Transfer to the natural env’t

 The transfer of plantlets from in vitro to the ex vitro external env’t