Plant Tissue

Culture
 OBJECTIVES

• Introduction.

• Plant tissue culture.

• Methods of plant tissue culture.

• Applications of tissue culture.

 Introduction
• Tissue culture is of two types:

 Plant tissue culture .

 Animal tissue culture.

• Plant tissue culture is in- vitro

cultivation of plant cells or tissues.
 Definition

• Plant tissue culture –

Technique of growing plants cells, tissues, seeds and other parts in sterile environment
on nutrient medium.

• Growth of plants parts in vitro in nutrient media.

• Laboratory techniques of growing, culturing, maintaining cells, tissues in vitro.

 History

• Principles of tissue culture were involved 1838 – 1839 in cell theory advanced by
schleiden and Schwann.

• 1902- idea of totipotency of plant cell given by Haberlandt.

• 1926 – went discovered first plant growth hormone – Indonesia acetic acid.

• 1939 – Gautheret, White and Nobecourt establishes endless proliferation of callus culture.
 Plant Tissue Culture

• Collection of techniques used to maintain or

grow plant cells, tissues or organs.

• Under aseptic conditions on nutrient culture

media.

• Used to produce clones of plants .

• By process of micropropagation.
Basic Requirements Of
Plant Tissue Culture

• Plant material.

• Equipments and
Glassware.

• Nutrient medium.

• Proper , optimum aeration.

 Steps Involved In PTC

• Nutrient media preparation.

• Sterilization of nutrient media.

• Preparation of explant.

• Incubation.
Advantages & Disadvantages Of Plant Tissue Culture
Advantages

 Production of multiples of plants in absence of seeds.

 Disease free and desired propagation.

 Improvement of medicinal plant species.

 Growing of new plantlets in short period of time.

Disadvantages

Require more labor and cost more money.

Chance of less resilient to disease due to type of environment in which they grown.
 Principles Of Plant Tissue Culture

• Plasticity:

Ability of plant to alter metabolism, growth and development to best suit their
environment.

• Totipotency:

Ability of plant cells to regenerate into whole plant.

 Types Of Plant Tissue Culture

• Seed culture, Embyo culture, meristem

culture,Bud culture.

• Callus culture, cell suspension culture.

• Anther culture/ ovary culture.

• Leaves culture.
 Callus culture

 Cell division in explant forms a callus.

 Callus formation is stimulated by darkness and solid medium.

 Auxins and BAP(Benzyl amino purines).

 Callus is obtained within 2-3 weeks.

 Anther Culture
• Anthers are young flower buds removed from plant.

• Then excised and transferred to nutrien medium.

• After 4-5 weeks plantlets are formed.

• From single anther many plantlets are formed.

 Pollens culture

 Pollens are removed from anther.

 Placed in 5ml liquid medium in petri dish.

 Petri dish is sealed with parafilm and incubate at 28 C in dark for 14 days.

 3-4 weeks may required.

Leaves Culture

• Leaves Detached from plants and place in

healthy condition for long period.

• The growth potential is more in young

leaves as compared to nearly mature
ones.

• Growth rate depends on their stages of

maturity at excision.
 Single Cell Culture

 Earlier, cells derived from single cell through mitosis constitute clone.

 Process of obtaining clones is called cloning.

 There are two popular techniques for single cell culture.

 Bergmann’s plating technique and Filter paper Raft Nurse Tissue Technique.
 Endosperm Culture

• For culturing endosperm tissue culture methods

are also used.

• Techniques of endosperm culture are following :

 Initial callus phase is developed.

 Ex is ed endosperm are cultured on a

suitable medium and embryo are removed
after initial growth.
 Embryo Culture
• Culturing young embryos on a nutrient medium = embryo culture.

• From developed seeds young embryos are formed.

• Older embryos are more easily cultured in vitro than young embryos.
 Methods Of Plant Tissue Culture

• Two major methods

1. In vitro growth callus and suspension cultures.

2. Type of explant – single cell culture, anther culture, proptoplasm ulture, embryo
culture, ovule culture ovary culture.
Environmental Conditions

• Three important aspects in vitro culture:

1. Nutrient medium.

2. Aseptic Conditions.

3. Aeration of tissues.
 Nutrient Medium

• Medium depends on type of plant tissue or cell used.

• Nutrient medium Usually consists of :

• Inorganic salts, a carbon source,vitamins and growth

regulators.

• Amino acids and pH of 5.7.

 Aseptic condition

• Tissue culture Should be done In completely

aseptic condition.

• Should be done so that that unit and

equipments are free from microorganisms.

• Some methods are used :

• Dry heat, wet heat, ultrafiltration and

chemicals.
 Aeration Of The
Tissue

 Proper aeration is important aspect

of culture technique.

 Achieved by occasionally stirring the

medium.
 Applications Of
Tissue Culture

• Rapid conclusion propagation

• Transgenic plants.

• Resistance to weedcides.

• Induction and selection of mutations.

 Media

• Food material or substances required for growing microorganisms in vitro.

• Media is growth medium or culture medium , solid, liquid or semi solid, support growth of
cells and tissues.

• Either organic or synthetic substance.

• Provides both biochemical and biophysical factors necessary for growth of bacteria.
 Functions of Medium

 Provide vitamins, water,minerals.

 Provide growth regulators.

 Removal of plant metabolite waste.

 Plant growth factor pic

• Light, games.

• Temperature.

• Water, humidity.

• Nutrition and hormones.

 Plant Growth Regulators
• Plant hormones can be defined as small organic molecules that elicits a physiological
response at very low concentration.

• Act as messenger between en Ironman and genome.

• Plays an important role in the phenotype.

Functions Of Growth Regulators

• Auxin-

Promote roots growth and cell division.

• Cytokinin-

Promote shoot growth and cell division.

• Gibberellin-

Promote cell enlargement and shoot elongation.

• Ethylene-

Low concentration can promote process.

 Needs Of Media

• Microorganisms and cells need

nutrients, energy to grow and
reproduction.

• In laboratory the requirements for

growth and reproduction are met
by culture medium.

• That is why the nutrients are added

in medium.
 Ph Of Tissue Culture Media
• Ph is adjusted between 5 and 5.8 before gelling .
• Sterilize with help of dilute NaOH, KOH or HCL.
• Ph below 5 will not get properly.
• Ph above 6 may be too hard.
 • Dry heat treatment.

Sterilization Techniques • Flame Sterilization.

of Medium • Autoclaving.

• Wiping 70% ethanol.

 Sterilization Of Medium
• Culture medium may contain microorganisms so important to sterilize it.

• Culture medium usually sterilized in autoclave at 121 C.

• 15 psi for 20 minutes.

 Incubation Of Medium

Explants are incubation under controlled conditions of:

• Temperature and Humidity.

• Cultures are incubated for 3 to 4 weeks.

Preparation Of Nutrient Medium

 Preparation involves preparation of stock solutions (10x to100x).

 Using high purity chemicals and distilled water.

 Solution stored In glass or plastic containers.

 Use growth regulators.

 Haploid and
Micropropagatio
diplodocus

Tissue
n.
production.

culture
Applications Germplasm Synthetic seed
preservation. production.
 Micropropagation

• Art and science of plant multiplication in

vitro.

• Large scale production of genetically

identical clones .

• Derived from meristems or vegetative buds.

• Practice of rapidly multiplying stock plant

material to produce large number of
progeny.
 • Easy to manipulate production cycles.
Features of • Disease free plants can be produced.
Micropropagation
• Clocal reproduction.
 Steps Of Micropropagation

• Stage 0.

• Stage I.

• Stage II.

• Stage III and stage IV.

 • Selection of explant.

Stage 0-I • Surface Sterilization.

Establishment • Washing.

• Establishment of growth medium.

Stage II- • Transfer to proliferation media.
Multiplication • Shoots can be constantly divided.
  Transferred to root media.
Stage III- Rooting
and Hardening  Explants returned to soil.

 Hardening Of shoots to increase strength.

 Methods of • Organogenesis.
micropropagation
• Embryogenesis.

• Micro cutting.
 Organogenesis
• Process of morphogenesis involving formation of plant organs.

• It is of two types:

1. Direct organogenesis.

2. Indirect organogenesis.
 Direct organogenesis

• Directly culture of tissues from leaves, stems, roots etc.

• The tissues undergoes morphogenesis without going through a callus culture.

• And without going through suspension cell culture stage.

Indirectly organogenesis

• Organogenesis occurs through callus and

suspension cell culture formation.

• Callus growth canbe established from many

explants.

• From leaves, roots, stems, flowers etc.

 Embryogenesis

• Process of regeneration of embryo from somatIc cells, tissues or organs is referred as

somatic embryogenesis.

• Somatic embryogenesis is of two types :

1. . Direct somatic embryogenesis.

2. Indirect somatic embryogenesis

 Direct somatic embryogenesis
• When embryos develop directly from cell or small group of cells.

• Such as styles or pollen.

• Without formation of callus.

• It is generally rare.
 Indirect Somatic Embryogenesis

• Cells from explant are made to proliferate and form callus Tissues or from cell
suspension culture.

• It is possible in presence of growth regulators.

• In presence of suitable environmental conditions.

 • Produce disease free plants.

• Production of many plants that are clones

Advantages of of each other.
Micropropagation • Maintain genetically uniform progeny.

• Easy and economical method of plant

propagation.
Disadvantages of
Micropropagation

• Expensive laboratory equipments and service.

• An Infected plants sample can produce infected

progeny.

• Relatively expensive to set up.

• Contamination of culture.
Application Of Micropropagation

• Rapid increase of stock of new varieties.

• Cloning of planttypes not easily propagated by conventional methods.

• Elimination of diseases.

• Cost Effective process.

Micro- Cutting

• It is of two types:

• Meristem culture( Mericloning).

• Bud culture.
 Meristem Culture
• Cultivation of axillary or apical meristems.

• Involves development of already existing shoot meristem.

• Produces virus free callus.

• It has capacity of producing a complete plant in vivo and in vitro.

 Bud culture

• Surface sterilization in NaOCL.

• Rinsing with SDW.

• Transfer into sterile container.

 Shoot tip culture

• Culture of terminal portion of shoot (0.1- 1.0mm).

• Comparising meristem(0.0- 0.1mm).

• With primordial and developing leaves and adjacent

stem tissues.
 Germplasm Conservation
• Germplasm is collection of genetic resources for organism.
• Germplasm may be stored as a seed, stem, callus, whole plant in nurseries in plants.
• It is a genetic source material in form of seeds, cultured cells, callus.
• In situ/ex situation preservation of these material is called Germplasm conservation.
 Methods of
Germplasm

In- situ preservation. Ex- situ preservation.

 • Preservation in natural habitat.
• Conservation of domesticated and
cultivated species in surrounding.
• There is high loss or decline of
species.
• Lead to loss of biodiversity.

In – situ preservation
 Disadvantages Of In situ preservation.

• Cheap and convenient way of conserving biological diversity as we play supportive role
only.

• Genetic diversity may have already been dramatically decreased.

• Threatened conditions may still be present in this area.

 Ex- situ preservation

• Maintain biological material outside their

natural habitats.

• Means literally Off site conservation.

• Particularly important for some

endangered species.
 Loss of seed viability.

Poor germination rate.

Disadvantages
Of Ex- situ
Conservation
Costly process.

Only useful for seed propagating plants.

 Explant
• Any part of plant taken out and grown
in test tube under sterile conditions in
specific nutrient media is called explant.

• Plant tissue cultures are generally

initiated from multicellular tissue
fragments called explant.

• tissues of Leaf, stem, root, hypocotyl,

embryo are used to originate explant.
 Preparation Of Explants

• Transferred explant tissue from plants to nutrient medium.

• Explant may be taken from any part of plant.

• Age of explant.

• Homozygous plants are preferred.

 Selection Of Explant

• Explant is selected either haploid or diplodocus explant.

• Plant growth achieved in two ways:

• By somatic embryogenesis.

• By Shoots directly.
 Polyploid

• Organism has more than two complete sets of chromosomes.

• Polyploids divided into many types:

• Triploids, tetraploids, penta, hexa,octa, deca, dodecaploids.

Importance Of Polyploids

• Self fertilization.

• Create new plant species and genotype.

• Identify genetic origin of crops.

• Help in obtain seedless fruits.

 Mutation

• Inheritance changes in characters of organisms.

• It can be beneficial or harmful.

• Occur naturally or by experimentally.

Importance Of • Used to produce different traits in crops:
Mutation • Large seed, new colors, sweeter taste.

• Agricultural revolution.

• Producing new species of crops.

 Cryopreservation
• Cryopreservation is storage of biological material at ultra low temperature.

• At temperature of liquid nitrogen all metabolic activities of cells are ceased and
preserved.

• Process of preserving or storing cells, tissues, organs or any other biological materials.

• At very low temperature( - 80 C using CO2 or – 196 C using liquid nitrogen.

Requirements for Preculturing.
Cryopreservation
Cryoprotection(glycerol, PEG protect
from ice damage).

Freezing:

Storage.
Principle of Cryopreservation

Cryopreservation is based on conversion of water present in cells

from liquid to solid.

Metabolic processes and biological divisions in the cells are almost

stopped when stored at low temperature.
 Classical cryopreservation

• Cooling is performed in presence of ice.

• They are generally operationally complex.

• Cryopreservation following classical protocols induces a freeze dehydration process.

• It involves cryosotection by using different cryoprotective solutions combined or not with

Pregrowth Of material and followed by slow cooling.
 Plant species obtained using tissue
culture

• Artemisin is derived from plant source Artemisia spp used as

Antimalarial.

• Codeine derived from papaver spp. Uses as Analgesic.

• Quinine from cinchona officinalis used as antimalarial.

Thank You