Chapter II.

Plant tisssue culture

2.Plant tissue
culture
Introduction

• What is plant tissue culture?

• When did it begin?
• Why we need tissue culture facilities ?
• What basic facility we need in tissue culture lab?
• Tissue culture started with discovery of cells

• The most significant success in plant tissue culture

was the formulation of a defined culture medium
(Murashige and Skoog 1962).
Plant tissue culture: the growing
and multiplication an explant (plant
cells, tissues, or organs etc…) on
• specially formulated nutrient
media
• under aseptic and suitable
growth condtion
• usually in a glass container
 Terminologies
• Asepsis: refers to growth of plant tissues under
conditions free of microbial contamination

• Totipotency: is the concept that a single cell has the

genetic program to grow into an entire plant

• An explant: is the piece of the plant (propagule)

used to initiate the tissue culture process

• Tissue culture is sometimes referred to as ‘sterile

culture’ or ‘in vitro’ culture.
Why we need tissue culture?

• Plants usually reproduce through

sexual means
• Flowers and seeds to create the next
generation

• Some plants and trees take several

years before they flower and set
seeds making plant improvement
difficult and time consuming

• Tissue culture technology

contributes significantly to the
improvement of agricultural
productivity and food security
Why we need tissue culture?

▪ Very fast technique: Thousands of plantlets can be produced in a few

weeks time from a small amount of plant tissue.
▪ Disease free plant can be produced
▪ Grow plants round the year irrespective of weather or season.
Very little space is needed for developing new plants by tissue culture.
▪ Helps to speed up the production of new varieties into the market place.
Helps in maintaining and establishing virus free stock.
▪ So, we have understood that tissue culture is a technique which is
important for transforming plants with new genes.
2.2 Requirements for a plant tissue culture laboratory

The physical components of a typical plant tissue

culture facility include:

• Correctly designed Buildings: with preparation room,

transfer room, culture or growth room, hardening area
(greenhouses, plastic tunnels), packaging and shipping
area, and related facilities office and store for
chemicals, containers and supplies

• Laboratory equipment, facilities , reagents

Correct location and design of a laboratory
Properly designed laboratory:
• Reduce contamination, achieve a high efficiency in work
performance
• Can reduce both the operational and energy costs

• Location: check out the area keeping in mind:

• access to water, electricity, transportation, and infrastructure for

supplies.
• Away from high traffic areas
• connection to septic system

How large :
• The minimum area required for media preparation, transfer and
primary growth shelves is about 14 m2.
 Design and lay out of the laboratories

• Correct laboratory
location
• Access to water
and electricity
• Isolation from trafic
• Connection to
septic system
• Suitable design
• To creat and
maintain asepsis
A sketch plan for setting up a Plant tissue
• To increase
culture laboratory. working efficieny
• Floor : Concrete
• Walls and ceiling should be insulated and covered inside
with water resistant material
• Windows, if desired, may be placed wherever convenient in
the media preparation and glassware washing rooms.
• The heating system should be capable of maintaining room
temperature at 20 degree centigrade during the coldest
part of winter
• Connection to a septic system or sanitary sewer
• Air conditioning requirements should carefully estimated
• Electrical service
• capacity for equipment, lights and future expansion
Growth room
• The cleanest rooms or
areas are the culture room
(aseptic transfer area) and
growth room
• There should be no direct
access to these rooms
from outside
Preparation Room Transfer Room
1. Autoclave 1. Laminar flow cabinets
2. Water distillation and sink unit 2. Bench and presses
3. Hot plate stirrer 3. Peristaltic pump
4. pH meter 4. Chairs
5. Weighing balance 5. Safety burner/sterilizers
6. Weighing balance 6. Gas cylinder
7. Hot-air oven 7. Binocular microscope
8. Microwave 8. Gyratory shaker
9. Refrigerator
10. Cupboards and presses
11. Trolley
• Washing Area
• The washing area should contain large sinks, some
lead-lined toresist acids and alkalis, draining boards,
and racks, and have

• access to demineralized water, distilled water, and

double-distilled water.

• Space for drying ovens or racks, automated

dishwashers, acid baths, pipette washers and driers,
and storage cabinets should also be available in the
washing area.
Water sources :
• The water used in preparing media must be of the
utmost purity and highest quality.

• Tap water is unsuitable because it may contain cations

(ammonium, calcium, iron, magnesium, sodium, etc.),
anions (bicarbonates, chlorides, fluorides, phosphates,
etc.), microorganisms (algae, fungi, bacterial), gases
(oxygen, carbon dioxide, nitrogen), and particulate
matter (silt, oils, organic matter, etc.)
• Media preparation and strilization room
• Central room, most activities performed here
• Autoclave
• pressure cooker
• pH meter
• fridges, stirer
• microwave oven
• A refrigerator/ freezer to store chemicals and stock
solutions
• Weighing scales
• Bench space for hot plates/stirrers, pH meters,
balances, water baths, and media-dispensing
equipment should be available
Certain spores from fungi and bacteria are killed only at 1210C and
1.05kg/sq.cm (15 pounds per sq. inch) pressure

Self-generating steam autoclaves are more dependable and faster to

operate.
• Aseptic transfer room
• Enable to tranfer sterile explant to sterile media under
sterile condtion
• The laminar flow chambers
• Bunsen burner/glass-bead sterilizers
• Microscopes

• The laminar flow chambers provide clean

filtered air that allows cultures to be
handled under contamination-free environment.
• Success of PTC depends entirely on:
• Creating and maintaing asepis
• Quality of media prepared for a given
plant tissue/techniques
• Using an apropriate explant
Principles and methods of aseptic
culture
• What mean when we say aseptic condtion?
• What are the sources of contamination?
• What are the most contaminant microorganisms
• How to creat and maitain aspetic culture in plant
tissue culture Laboratory?
• Aseptic technique: Common laboratory procedures
that can reduce the risk of culture contaminations

• Two major strategies of aseptic work are described:

using a Bunsen burner and a laminar flow hood

• Aseptic technique is a set of routine measures that

are taken to prevent cultures, sterile media stocks,
and other solutions from being contaminated by
unwanted microorganisms (i.e., sepsis)

Asepsis simply implies freedom from pathogenic

organisms
Examples of aseptic technique are

• Cleaning and disinfecting lab surfaces prior to use

• limiting the duration that cultures or media are
uncapped and exposed to the air
• keeping petri dishes closed whenever possible
• effectively sterilizing inoculating loops and other
equipment that comes into contact with cultures or
media ,and
• avoiding breathing on cultures or sterile instruments.
The critical areas of concern with respect to successful
aseptic technique include:
❖ environmental conditions (laboratory or work space),
❖source material (cell lines, media, and reagents),
❖equipment (labware, instruments, and apparatuses)
❖sterilization procedures and equipment (autoclave,
dry heat, filtration), and
❖human (laboratory personnel) considerations.
• Sterilizing Equipments and Reagents
• Using Filter sterilization
• heat-labile substances
• Physically removes contaminates
• Autoclaving
• Typical autoclaving conditions of 121◦C
(250◦F) for 15 minutes at 103 kPa (15 psi) are
sufficient to kill virtually all forms of life,
including bacterial endospores, and will
inactivate viruses.
Cleaning and disinfecting the space inside hood
(after use)

• When finished using the hood, remove all unnecessary

items, and then clean all surfaces by liberally spraying
with 70% alcohol.

• Turn on the UV light at the end of the work day to help

keep down levels of potential contaminants
• A laminar flow unit (or hood) is
a sophisticated appliance that
can further help prevent
contamination of reagents and
biological cultures.

• Benches for aseptic work should

be organized away from air
conditioning vents, cooling fans,
open windows, or blowers from
heating or refrigerating systems
• Most contaminat mos in TC lab.
• Bacteria most important
• fungi
• virus
• micoplasma, yeast
• How they affect growth and development?
• They decrease the PH of the media
• Competing for resources
• Releasing phytotoxic chemicals
Method to creat sterile
condtion
Sterilization
• Steam sterilization (media,
culture vessles, small
Instrument)
• 121 0C temperature
• 15 kpa presure
• For 15 min

• Proccedure to maintain
asepsis
• Preparations of sterile
container, media, small
instrument
• Success of wet heat sterilization depends on
• Volume of the object to be sterilized
• Time
• Presure and temperature
• Cover small instument with alluminium sheet to
prevent contamination after autoclaving

Dry heat oven

• Commonly used to strilize metal instument and glass
vessle
• Dry oven is used at temp. 160 to 180 0C
Surface sterilization of explant
It must be free of MOs
• Wash in tap water
• Deep in 70% ethanol
• Immerse in 1-5% of NaoCl, CaoCl (2 to 20 min)
• Wash with sterile distilled water 3-4 times to remove any
traces of NaoCl

• Most commonly used sterilant

• Na & Ca hypochlorite (1-5%)
• Ethanol 70- 90%
How to maintains aseptic condtion
1. Clear and disinfect the bench of the laminar-flow hood
working surface and the left and right sides of the hood with
70% ethanol or other disinfectant.

2. Turn the hood blower and lights on and let the air circulate
within the hood 10 min before use.

3. Place items needed for the specific procedure into the hood,
wiping each item with 70% ethanol or other disinfectant just
before introducing it into the laminar environment.

4. Wash hands well before working in the hood and wear a clean
laboratory coat and surgical gloves to further protect the work
from shedding of skin flora that can contaminant any product
5. While working in the hood, and avoid rapid movements,
equipment that might disrupt the laminar air flow.

• Perform all work at least 10 cm back from the front opening

of the hood and avoid moving materials or hands in and out
of the cabinet as much as possible.

6. If flame sterilization is needed in the hood for a particular

application, use a burner/glass

7. After work is completed, remove all material from the

laminar work bench, clean any spills, and disinfect the bench
working surface by wiping with 70% ethanol or other
disinfectant.

8. Turn off hood blower and lights.

• Materials used to avoid recontamination
• Antibacterial soap, 70% ethanol or other
appropriate disinfectant
• 95% ethanol
• Clean laboratory coats or gowns
• Latex surgical gloves
• Clean, quiet work area
• Shallow discard pans containing disinfectant Bunsen
burner or pilot-activated burner
Tissue culture medium Components
• What is tissue culture media?
• Why we need them?
• What are the major type of media
• What are the components of media
• What are the steps in media propagation
• A growth medium or culture medium is a solid, liquid or
semi-solid designed to support the growth of
microorganisms or cells or small plants

• Plant grown in the field get nutrients, air and sunlinght from
the environments.

• In vitro growth of plants is largely determined by the

composition of the culture medium and growth condition

• The main components of most plant tissue culture media are

mineral salts and sugar as carbon source and water.

• Other components may include organic supplements,

growth regulators, a gelling gent.
Selection of Media
• A nutrient medium consists of all the essential major and minor plant nutrient elements,
vitamins, plant growth regulators and a carbohydrate as carbon source with other
organic substances as optional additives.

• Components of media can be classified into five groups:

1. Inorganic nutrients
• Macronutrients
• Micronutrients
2. Organic nutrients
3. Carbon source
• sucrose most common (3%)
• Household sugar and other sugar
4. Solidifying agent
▪ Agar is most commonly used for preparing semisolid or solid culture media
▪ Other gelling agents are occasionally used including gelatin, agarose, alginate and gelrite
5. Growth regulators
• Hormones are organic compounds naturally synthesized in higher plants which influence
growth and development
• There are two main classes of growth regulators used in tissue culture, auxin and
cytokinins
PTC media components

• Inorganic minerals (macro and micro), N, P,K Mg, Ca,

S. Cl Mn, Co, Zn, Mo, Fe.
• Organic supplements (vitamins, Amino acid, and
carbon source)
• The amount of these substances required for
successful culture varies with the species and
genotype, and is probably a reflection of the
synthetic capacity of the explant.
Major types of media

• The basic media formulation that are most

widely used media.

✓ MS (Murashige and Skoog, 1962) and

✓LS (Linsmaier and Skoog, 1965)

✓Woody Plant Medium (WPM)
• Media compositions have been formulated
for the specific plants and tissues

• Amounts of the various ingredients in the

medium vary for
➢ different stages of culture and plant species
➢ Purpose of the culture
• Tissue culture media
• An artificial nutrient culture medium components
have been investigated for several crops because
of the complex nutritional and hormonal
requirements each genotypes/species needs

• A key advance in plant tissue culture was the

control of morphogenesis by using different levels
and combinations of growth regulators
• Gelling agent : added to the culture medium to
increase its viscosity as a result of which plant tissues
and organs remain above the surface of the nutrient
medium.
• agar, ‘Agarose’, and ‘Gellan gum’
• The lowest concentration of agar, which can be used,
depends on its purity and brand.
• Agar is usually used at 0.6-0.8% (w/v).
• Agar brands vary widely in price, performance and
composition
• Conventional carbon sources and low cost alternatives
• Sucrose is the most commonly used carbon source in
the micropropagation of plants, but expensive
• Household sugar and other sugar sources can be used
to reduce the cost of the medium
Source of water
• Distilled or doubled distilled and de-ionized water is
used
• If tap water is free from heavy metals and
contaminants, it can be substituted for distilled water.
• Plant growth regulators ( auxin and cytokines, gibberellins)

• Most commonly used PGRs

• Affect growth and development of cultured cell
• Their relative ration determines morphogenesis
of the plant

• High Auxin :cytokines - promote root initiation

• Low “ -promote shoot differentiation

The ratio of auxins to cytokinins in the culture

medium is important since their combination
determines the morphogenic response for root and
shoot formation.
• The ratio of auxins and cytokinins play an important role in
the morphogenesis of culture systems.

• When the ratio of auxins to cytokinins is high, embryogenesis,

callus initiation, and root initiation occur

• For axillary and shoot proliferation, the ratio of auxins to

cytokinins is kept low

• Among the gibberellins, gibberellin A3 (GA3) is the most

commonly used for tissue culture.
• GA3 enhances callus growth and induces dwarf plantlets to
elongate.

• What do you understand from the hormonal balance in the

nutrient media?
The nutrients in the growing media, sucrose, mineral salts, vitamins and growth regulators, are far in excess of those
in the soil
Steps to prepare MS media from commercially available
package
1. Measure 600 to 750ml
2. Add the recommended component of the package
3. Add carbon source, sucrose (30gm/lt) if not found in package
4. Add the required amount and type of the PGRs
5. Bring the volume to 1000ml by adding distilled water
6. Adjust the pH to the required level (5.8)
7. Add agar 8gm/lt
• Dispensing and then autoclaving
• Autoclaving, cooling and the dispensing to Petri-dish
Stock Solutions preparation and handling

• Usually the stock solutions are prepared in 10x to 1000x concentrations.

• The stock solutions consist of groups of chemicals, e.g. macronutrients,

micronutrients, vitamins and plant growth hormones.

• The use of stock solutions

• Reduces the number of repetitive operations involved in media preparation
• Reduces the chance of human or experimental error.
• Easy measurement specially in (micronutrients and hormones)

• Moreover direct weighing of media components (e.g., micronutrients and

hormones) that are required only in milligram or microgram quantities in the
final formulation cannot be performed with sufficient accuracy for tissue culture
work

• For these components, preparation of concentrated stock solutions and

subsequent dilution into the final media is standard procedure
Selection of sources of an explant
• Some species or even clones are easier to grow in culture than
others. Some respond reluctantly to culture, some do not
respond at all, and many plants have never been tried so far.

Why?

• Different genotypes will accumulate different endogenous

concentrations of growth regulators and the sensitivity of
different processes to these regulators will also vary with
genotype and with the status of other variables.
 Surface Sterilization
The procedure vary depending on: sample type,
sources and other. Each plant species needs specific
sterilization procedure however, the main
procedures includes.
• Wash samples gently under running tap water to
remove any dust particle
• Samples are drained of tap water and dipped in
75% Ethanol for specified seconds or minutes
• Submerged in sodium or Calcium chloride with
specific concentration solution ( like 2-10% )
• Rinsed three times with distilled water for 3, 10,
20 minutes
 Culture containers
• A wide variety of culture containers are available on the
market.
• The plant performance and cost of the containers decide
which to choosing the appropriate

• Depending upon the scale of production, different types of

containers are deployed for culture initiation, maintenance of
mother cultures and sub-culture for multiplication.

• For shipment of plants: disposable containers should be

bought
• For maintaining in vitro plants: containers should be
transparent to facilitate illumination and easy inspection.
Types of culture containers

• Glass test tubes have been universally used for culturing

plant tissues as their narrow openings keep out
contamination
• 1 to 2 explants are cultured in each test tube
• As explant growth and multiplication progress, the cultures
are transferred to larger containers.
• Test tubes should not be used for large-scale multiplication of
plant material.
• Conical flasks, 150-250 ml capacity, are mostly used for the
micropropagation of plants in liquid media, and are kept on
shakers for agitation of the medium and suspension cultures
• Plastic bags are sterile due to high temperature during
manufacture.
• Glass and plastic Petri dishes are also used to culture explants,
which are then transferred to larger vessels for multiplication and
elongation.

• Pre-sterilized disposable-plastic Petri dishes are much cheaper. In both

cases, medium is poured under the laminar flow hood.

• Glass bottles and baby-food jars with polypropylene caps are the
most widely used containers and the most economic and low cost
option.

• Such containers can be washed, sterilized and reused repeatedly.

• Transparent plastic containers, such as ‘Magenta’TM vessels, that

withstand autoclaving and washing, are extensively used for
micropropagation in many developed countries,
Vessel closures and lids
• The vessel closures influence growth of the in vitro plants

• The culture containers have to be closed to keep out microorganisms but they should not be
sealed so tightly that gaseous exchange is prevented.

• Different types of closures includes

• non-absorbent cotton plugs,
• Polyurethane foam plugs,
• specially made plastic plugs,
• aluminum foil,
• stainless steel caps,
• Polypropylene caps with bends,
• PVC film, Polythene film and silicon rubber.
• Explant: different explant type can be used
depending on the plant species and objectives to
be attained
2.5 Plant Tissue Culture:
methods and Applications
• Plant tissue culture is a significant contribution
✓in micropropagation of ornamental and
forest trees,
✓production of pharmaceutically
interesting compounds and
✓ Plant breeding for improved nutritional value of
staple crop plants as well as in the improvement of tree
species
Plant tissue culture can provide

• High-quality planting material for the fruits,

vegetables and ornamental plants and forest tree
species throughout the year, irrespective of season
and weather, thus opening new opportunities to
producers, farmers and nursery owners.

• The biotechnological approaches like haploid

induction, somaclonal variation, etc. to improve
traits are also its important applications.
• Plant tissue culture has been routinely done for
conservation of germplasm to be used for
improving secondary metabolite production and
agronomic traits of crops to increase yields.
 Micropropagation
• Micropropagation: is one of the most useful aspects of
plant tissue culture technique

• The process of micropropagation involves the following

four distinct stages

• The first stage is culture initiation

• Which depends on explant type or the donor plant at the time
of excision.

• Explants from actively growing shoots are generally used for

mass scale multiplication
• The second stage is shoot multiplication which is
crucial and achieved by using plant growth
regulators (PGRs) generally, auxins and cytokinins.

• In the third stage, elongated shoots are

subsequently rooted either ex vitro or in vitro.

• The fourth stage is acclimatization of in vitro grown

plants, which is an important step in
micropropagation
Organogenesis

• In plant tissue culture, Organogenesis, can be

defined as the ‘genesis’ or formation of organs from
unusual parts (i.e. adventitious development of
organs)

• In normal in vitro conditions and under the

influence of various factors, organogenesis is a two-
step process where shoots develop first and roots
next, giving rise to a complete plantlet.
• The adventitious origin may be attributed to either
direct differentiation of cells and tissues (explants)
to form an organ or via cells undergoing cycles of
dedifferentiation (caulogenesis) and
redifferentiation
Why driving force of dedifferentiastion
• Reorgnization in chromatin structures that
leads to expression of pluripotent genes.

• This can inturn brought by cell wall

removal, viral infection

• Varoius growth factors

• Certain internal and external factors determine
organogenesis.

• Internal factor mainly includes

• Genotype and endogenous levels of growth regulators.

• External factors
• explant type, season of explant harvesting and culture
room conditions (temperature, light, humidity, etc.) play
pivotal role in overall development of cultured plants.
Plant can be initiated from any one of
the following:
Callus culture
• Callus is disorganized losely
arranged mass of
paranchma cells

• Invitro, callus intiated and

formed under condtion of
high auxin to cytokines
ratio.

• Plant cells are totipotent

Callus culture…

• 2, 4, D the most important

callusing hormones in low
concentration

• IAA, IBA, NAA can also used

• Callus can varies interms
of color (creamy , yellow,
white and pigmented)
based on the type of
explant and environment

• Must cultured in dark at 25

0C
• Pigmented under light
Callus culture…

• Callus culture follow three developmental

stages
• Induction/initiation
• Dedefferentiation (into meristematic cells) and
intiation of cell division
• Cell division (rapid increase in cell number)
• Defferentiation
• Stationary stage, stage of sub-culturing
Sub-culturing: is the transfer of cell/ tissue /organ or
plantlets from old exhausted medium to a new (fresh
medium)

Why sub-culturing ?
• Exhauting of mineral nutrient from the media
• Media may became dry (evaporation of water, utilized by
explant)
• Accumulation of phytotoxic chemicals (waste get ride of by
the mass of cell)
• Densities of cells, tissue, organ becomes excesive
• To increase number of organ for micropropagation
Important application area of
• Variety development
callus culture
• Micropropagation
• Cell suspenssion
• Secondary metabolite
culture
prodution
• For cell/plant
• phenolic compound, plant
transformation/transg
hormones
enic plant production
• Somatic embryo prodution
• Production of
dihaploid plant
Organogenesis and somatic embryogenesis
• Organogenesis and somatic embryogenesis are the two
alternative pathways in plant tissue culture techniques for
propagation in addition to direct shoot induction.

• Organogenesis involves inducing the vegetative tissue to form

organs (shoot or root) which eventually develop into a
complete small plant

• Somatic embryogenesis is to induce a piece of somatic

(vegetative) tissue to develop an embryogenic callus, leading to
the formation of a somatic embryo which germinates into a
complete plantlet
• The original plant part used to initiate the culture is known as
explant.
 Direct and indirect Organogenesis
• Through the manipulation of plant growth
regulators and culture conditions, the explant
may develop shoot and root directly. This is
called the direct organogenesis.

• If the formation of shoot and root need to go

through a callus phase then it is called the
indirect organogenesis
Direct and indirect somatic embryogenesis

• Embryos can be developed directly from the explant

which is direct somatic embryogenesis

• When an embryo develop via a callus phase it is

indirect somatic embryogenesis.

• In the induction of somatic embryogenesis, direct or

indirect, auxins are used at the initial stage and
abscissic acid may be needed at a later stage
Organogenesis
• Can be achieved by direct and indirect methods
• Explant >>> organ
• Explant >>> callussing >>> organ

• Three methods of plant regenaration via organogenesis

• Prodution of adventitious organ from callus
• Emergence of adventitious organ from explant W/O callus
formation
• Production of plant from outgrowth of axilary buds
The prinicipal factor for organogenesis
• Sugested factors
• Stimulating effects of media components
• Endogenous compound in the culture
• type of plant
• Type and amount of auxin and cytokines in the media
SOMATIC EMBRYOGENESIS
• Somatic embryos can either differentiate directly from the explant without any intervening
callus phase or indirectly after a callus phase
• Explants from which Direct embryogenesis is most likely to occur include microspores
• ovules, embryos
Plant regeneration via somatic embryogenesis
includes five steps:

1. Intiation of embryogenic cultures : culturing the

primary explant on medium supplemented with
PGRs, mainly auxin but often also cytokinin.

2. Proliferation of embryogenic cultures: on

solidified or in liquid medium supplemented with
PGRs, in a similar fashion to initiation.

3. Pre-maturation of somatic embryos in medium

lacking PGRs; this inhibits proliferation and
stimulates somatic embryo formation and early
development.
4. Maturation of somatic embryos by culturing on medium
supplemented with ABA and/or having reduced osmotic
potential.

5. Regeneration of plants on medium lacking PGRs.

Developmental stages of somatic embryo

• Pro embryo: Small clusters of meristematic cells from which

somatic embryos will arise

• Globular stage: larger groups of cells not yet having a definite

embryo-like shape,

• Heart stage: a characteristic three-lobed stage

where cotyledonary initials are separated from the
root pole,

• Torpedo stage: an elongated form of the heart shaped embryo,

• Plantlet: discernable small ‘seedling’ with primary

root and shoot.
Factors affecting Somatic Embryogenesis
• Growth hormones

• Genotype of explants

• Concentration of some other substances like sucrose and

maltose
Applications of Somatic Embryogenesis

• Clonal propagation of genetically uniform plant material

• To produce haploid embryo by culturing anther/pollen/ovule

• Synthesis of metabolite.

• In genetic transformation it provides source tissue

• Synthesis of artificial seeds (for micropropagation purpose)

• Generation of whole plants from single cells called protoplasts

• To obtain Somaclonal variant embryo

Micropropagation

• Micropropagation starts with the

selection of plant tissues (explant)
from a healthy, vigorous mother
plant

• Any part of the plant (leaf, apical

meristem, bud and root) can be
used as explant
What is Micropropagation?
• Micropropagation is the growing of plants from meristematic tissue or somatic
cells of superior plants on nutrient suitable media under controlled aseptic
physical conditions.

• The true to type propagation of the selected genotypes using in vitro techniques.
Advantage
• The primary advantage of micropropagation is the rapid production of high
quality, disease-free and uniform planting material

• Plants can be multiplied under a controlled environment, anywhere, irrespective

of the season and weather, on a year-round basis.

• Production of high quality and healthy planting material of ornamentals, and

forest and fruit trees
• Global trading for producers, farmers, and nursery owners, and for rural
employment
Why we do micropropagation

• To multiply plants whose multiplication rate is very

low
• To produce progenies which are genetically identical
to their parents
• Recovery of distant hybrids
• Germplasm conservation
• Germplasm exchange
• Genetic transformation – addition or deletion of gene
Cost of micropropagation
• Micropropagation technology
• is more expensive than the conventional methods of
plant propagation
• requires several types of skills.
• It is a capital-intensive industry, and in some cases the
unit cost per plant becomes unaffordable
• How to overcome the problem
• inventing reliable and cost effective tissue culture
methods without compromising on quality
• Needs constant monitoring of the input
costs of chemicals, media, energy, labour and capital
• Labour is account most cost of micropropagtin
developed country
• Indeveloping country: For example, the cost of
medium preparation (chemicals, energy and
labour) can account for 30–35% of the
micropropagated plant production.
Techniques of Micropropagation

• Micropropagation by axillary and apical buds

• Micropropagation by axillary shoots (buds, bulbs and
protocorms)
• Micropropagation through callus culture
• Artificial seeds
• Somaclonal variations
• Micropropagation, in combination with radiation-
induced mutations, speeds up the recovery,
multiplication and release of improved varieties in
vegetatively propagated plants.
Stages of Micropropagation
The process of micropropagation is
usually divided into several stages
i.e., pre-propagation, initiation of
explants, subculture of explants for
proliferation, shooting and rooting, and
hardening.
Selection and maintenance of stock
plants for culture initiation
In practice, the “explant” is removed surgically, surface
sterilized and placed on a nutrient
medium to initiate the mother culture, that is multiplied
repeatedly by subculture.

Extensively used in commercial micropropagation:

• Shoot-tip and meristem-tip culture:
• Nodal or axillary bud culture
• Floral meristem and bud culture
• Other sources of explants
• Cell suspension and callus cultures
Stage I. Initiation and establishment of aseptic culture

Surface sterilization
• Washing – Washed with water

• Establishment of explant on appropriate medium –

There is no one universal culture medium; however
modifications of Murashige and Skoog basal medium
(Murashige and Skoog, 1962) are most frequently used.
Stage II
Multiplication of shoots

• This stage, rapid multiplication of the

regenerative system is carried out for obtaining
large number of shoots on a defined culture
medium

• medium for stage I and II is same, but

cytokinin proportion is increased for stage II
to produce numerous shoots

• This stage can be repeated a few cycles until an

desired number of shoots are developed to carry
out for rooting

• Factors which can affect shoot multiplication

are physiological status of plant material,
culture media, culture environment.
• Number of subculture? Generally 10 to 12 to
reduce epigenetic variation and mutation
Stage III
Rooting of regenerated shoots

• In this stage, shoots or shoot clusters from stage II are

prepared to be transfer to soil.

• shoots are separated manually from clusters and transferred on

a rooting medium containing an auxin (in vitro root
development)

• Elongation of shoots prior to rooting, rooting of shoots

(individual or clumps), and pre hardening cultures to improve
survival are some of the activities carried under this stage.

• Sometimes, shoots are directly established in soil as micro-

cuttings to develop roots. (Ex vitro root development)
Stage IV
Transfer to the green house condition
(Hardening)

• Transfer of plantlets to sterilized soil

for hardening under greenhouse
environment.

• Most species require acclimatization

to ensure their growth and
development (involve physiological
and anatomical adjustment to adapt
field /green house environment)

• Increase survival and addaptation to

actual field condition
Procedure to Acclimatization of Micropropagated Plants
• In vitro rooted plantlets are washed with sterile water to remove
adhered nutrient agar and transferred to sterile soilrite in the
culture bottles.

• In case of ex vitro rooted plantlets after roots were visible, the

culture bottles were shifted from low temperature/high relative
humidity (RH) regime of green house to the region which
experienced relatively high temperatures and low RH.

• Also the rooted plantlets were exposed gradually to external

environment by loosening/removing the caps of the culture bottles.

• Micropropagated and hardened plantlets were transferred to

polybags containing mixture of organic manure, garden soil and
sandy soil.

• These plants were watered regularly. The green-house-hardened

plants were kept in nursery covered with agro net.
Advantage of micropropagation
• High multiplication rate per meristems/year
• Avoid probles of shoratge of plant
• Give chance to eliminate MOs
• Important for clonal propagation
• Not dependent on any seasonal change
• Helps to propagete seedless plants which are difficalt to
propagete by conventional methods
• Maintainamce and production of hybird matrials ex. Hybrid
cofffee
Limitation
• Costly
• Problem of manipulation of media
• Techniques for different plants are different
• Problems of somaclonal variation
Somaclonal Variation and Crop Improvement

Somaclonal variation: is variation that originate in cell and tissue

cultures, and the individuals are referred as “somaclone”

• Such types of variability among the cultures described to “off

type” or ”experimental error”

• Therefore, Somaclonal variation is general term used for the

variation detected among plants derived from any form of tissue
culture such as organ culture, protoplast culture and callus culture

• Show genotypic and phenotypic variations-

• Karyotype change (chromosomal number ) change in
chromosome structure, single gene mutation , sister-chromatin
exchanges, somatic genetic rearrangements, transposable
elements activity,
Origin of Variation
• Pre-existing genetic variation within the explants and
• Arises from (Chimeric) mother plant
• Variation induced during the tissue culture phase
• The continuous presence of higher doses of PGRs
influenced the mitotic activities which often induced
spontaneous variations in daughter cells
What are factors

• Growth media nature / culture condition

• Medium component particularly the growth regulators

type and composition highly influence

• Growth regulators like 2, 4-D, NAA, BAP are among the

important one to cause the chromosomal variability

• Sub- culture duration

Importance of Somaclonal variation

• Somaclonal variation is very important in plant breeding

program because it provide novel variation for plant
improvement that May not occur by conventional
breeding

• Variants possessing resistance to disease, insects and

tolerance to herbicides have been developed.

• In vitro selection shortens time in somaclonal isolation

with desirable trait
Disadvantages of somaclonal variation
• Serious problem in plant tissue culture industries as it
results in the production of undesirable plant
• Change is cultivar dependant
• May not be genetically stable and heritable and it
requires extensive field testing

To increase Somaclonal variation:

 Callus and suspension cultures for several cycles

 Regeneration of large number of plants from long-term
cultures
 Testing of selected somaclones for genetic stability
 Multiplication of genetically stable somaclones to
develop new cultivars
 Apical meristems Culture for Pathogen
Eradication
• What is apical meristems?
• Why it is possible to remove viruses
• What are the meachnism behind virus eradication?

• In addition to being used as a tool for plant propagation, tissue

culture is a tool for the production of pathogen-free plants.

• It is possible to produce disease-free plants by using meristem tip.

This technique is referred to as meristem culture, meristem tip
culture, or shoot tip culture, depending on the actual explant that
is used.
Meristem and shoot tip Culture
A meristem is a group of undifferentiated plant cells that can
divide and form all types of tissues.
Culturing of undifferentiated plant cells harvested from shoot-tips
- a technique used to get rid of plant viruses
• started first by Morel and co-workers in 1950s with the intention
of obtaining virus -free plants from those already infected
• 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
• Why meristematic tissues of roots and shoots of an
infected plant are sometimes free of viruses?

• The exact reason for this is not known, however, it is

believed that one or all the following factors are
responsible.

▪ High metabolic activity:

▪ Lack of vascular system
▪ High auxin concentration

• Since meristematic tissues are sometimes free of

pathogens, it is possible to recover non-infected plants
by in vitro meristem culture techniques and to grow
them into healthy plants
Techniques of meristem culture:

• Meristems are dissected out with the

• help of a sharp razor-blade ,
• the operation being carried out under a microscope

• single detached meristem could be cultured, divided into

several bits and each bit subculture.

• By repeating this several times over , an incredibly large

number of plantings could be obtained from a single protocol
in one year.
Invitro production of haploid plant
• The term haploid refer to plants possessing a
gametophytic number of chromosome ( single set) in
their sporophyte
• Important for production of homozygous plant

Haploid plant can be produced

o Spontaneously ( apomixes and parthenogenesis)
o Artificially (delayed pollination, application of irradiated
pollen, hormone treatment, temperature shock)
Haploid can be grouped
• Monohaploids: contain half the number of
chromosome from diploid spp. (Eg. maize and barely)
• Polyhaploids: ½ number of chromosome from
polyploidy spp. (Eg. potato, wheat)

Two ways to produces haploid plants

• Androgenesis: haploid production through anther
culture/microspore
• Gynogenesis: production of haploid plant from ovary
or ovule culture
Androgenic methods
• Haploid production from male
gametophyte of angiosperm plant
• Principle is to stop development
of pollen whose fate is normally
to became gamete and to force
its development directly in to
plant
Anther culture
Techniques
• Young flower buds with
immature anthers are
superficially sterilized and
washed with double distilled
sterile water.

• They are excised from the flower

buds and their proper
developmental stages are
determined under microscope.
• On confirmation of a
stage,
• the anthers are directly
transferred on nutrient
agar or liquid medium
where induction of
embryogenesis occurs,
or

• the pollen grains are

aseptically removed
from the anthers and
cultured on liquid
medium
▪ 10 to 20 anther can be planted on solid agar media in
6cm petri dishes

▪ 50 anther can be planted in 10 ml of liquid media

▪ Responsive anther , wall tissue turn brown with in 3 to

8 weeks and burst open due to pressure of growing
pollen callus or pollen plant

▪ The individual plants or shots are transferred to a

medium that would support further development

▪ The rooted plants are transferred to sterile soil mix in

pot
• Anther culture has advantage over microspore culture
because very quick for practical purpose and has aslo
conditioning effects of anther wall demerits (
influence development of microspore in it)

• For some spp the anther wall has inhibitory effects.

• For such species, microspore culture is a means to
overcome the problem

Advantage of microspore culture over anther

culture
o Over come inhibitory effects of anther wall
o The sequence of androgenesis can be observed from a
single cell
o Ideal for uptake, transformation and mutagenic studies
o Higher yield of plant per anther could be obtained
Factors governing the success of androgenesis
• Genotype of anther donor material

• Physiological status of the donor plant at time anther excision (

start with health pollen cell, avoid use of pesticide 2 to 4 weeks
preceding sampling, good growth condition ).
• Outdoor grown plant flower from young plant, are more
responsive

• Stage of pollen (anther with microspore ranging from tetrad to

binucleate stage are responsive)

• Pretreatment of anthers
• Cold treatment ( between 3&6 0C for 3 to 15 days) depend on spp
o Weak and non viable pollen are killed and vigorous anthers enriched
o Retards aging of anther wall & divert development from gametophtic
• Hot treatment ( 30 0C for 24hr stimulate embryogenesis, dissolution of
microtubule and spindle fiber)
• Chemical treatment
• Culture media
Composion of media determine success and mode of
development
Diplodiztion
• Haploid can be diplodized to produce homozygous
plant by the following methods
• Colchicines treatment
• spindle inhibitor to induce chromosome duplication
• Endomitosis :
• chromosome duplication with out nuclear division to form
diploid cell
• This is tendency of haploid cell in culture
• Significance and use of haploid plant

• Development of pure homozygous plant

• Main advantage is reduction in time to produce varieties
(6 to 8 years Vs few month to a year)
• Hybrid development (pure homozygous can be used to
produce F1 hybrids)
• Induction of mutation
• We can screen mutants resistant to disease , salt tolerance etc
• Induction of genetic variability
• ( by anther culture not only haploids but plants of various
policy levels and mutants are obtained and can be
incorporated in breeding program
 Germplasm Preservation
Cryopreservation: is the preservation of plant
genetic resources at ultralow temperature (−196 °C)
which enables plant genetic resources to be
conserved safely and cost-effectively

Cryopreservation
• Ultra low temperatures (-196 °C)
• Stops cell division & metabolic processes
• Very long-term (indefinite?)
Cryopreservation Requirements

• Preculturing
• Cryoprotection –Glycerol, PEG, to protect against
ice damage and alter the form of ice crystals
• Freezing –The most critical phase; one of two
methods:
• Slow freezing allows for cytoplasmic dehydration
• Quick freezing results in fast intercellular freezing
with little dehydration
Cryopreservation Requirements

• Storage –Usually in liquid nitrogen (-196oC) to

avoid changes in ice crystals that occur above -
100oC
• Thawing –Usually rapid thawing to avoid damage
from ice crystal growth

• Recovery -Thawed cells must be washed of

cryoprotectants and nursed back to normal growth
• Avoid callus production to maintain genetic
stability
 Embryo Culture
• Embryo culture developed from the need to rescue
embryos (embryo rescue) from wide crosses where
fertilization occurred, but embryo development did
not occur
Embryo Culture Uses

✓Rescue F1 hybrid from a wide cross

✓Overcome seed dormancy, usually with addition of
hormone to media (GA)
✓To overcome immaturity in seed
✓To speed generations in a breeding program
✓To rescue a cross or self (valuable genotype) from
dead or dying plant
 Embryo Rescue Process
• Make cross between two species
• Dissect embryo (usually immature)
–The younger the embryo, the more difficult to culture

• Grow on culture medium using basic tissue culture

techniques, use for breeding if fertile

• Many times, resulting plants will be haploid because of

lack of pairing between the chromosomes of the different
species
–This can be overcome by doubling the chromosomes, creating
allotetraploids
 Industrial Products from Cell Cultures
• Secondary metabolites produced by plants
–Alkaloids, Terpenoids, Steroids, Anthocyanins, Polyphenols
• Often unclear function in the plant

• Produced in specific: plant species, tissue or organ

• Many are commercially valuable

• Cell culture techniques allow large-scale production of

specific secondary metabolites