PLANT TISSUE CULTURE

METHODS & APPLICATIONS

By

OJUEDERIE Omena BernardDepartment of BiotechnologyCollege of Food SciencesBells University of Technology Ota.

IntroductionTissue culture “… A method of biological research in which fragments of tissues from

an animal or plant are grown in vitro

in artificial medium under aseptic

conditions and continue to survive and

function.”

IntroductionWhat is plant tissue culture?“… the aseptic culture of plant

protoplasts, cells, tissues or

organs under aseptic conditions

which lead to cell multiplication or

regeneration of organs or

whole plants.”

Basic concepts of plant tissue culture(PTC)

Two concepts, are central to understanding plant cell, tissue, organ culture and regeneration.

Plasticity.

-ability to initiate cell division from almost any tissue of the plant.

-ability to regenerate lost organs or undergo developmental pathways in response to particular stimuli.

Totipotency.

-each cell has the capacity to regenerate the entire plant.

Basic concepts of plant tissue culture(PTC)

Cells lines differentiate to form specialized tissues and organs.

Unlike animal cells, living plant cells re-differentiate.

Therefore, tissue can be regenerated from explants such as cotyledons, hypocotyls, leaf, ovary, protoplast, petiole, root, anthers, etc.

Short history of Plant Tissue Culture

A German plant physiologist Gottlieb Haberlandt (1902) cultured isolated single palisade cells from leaves in Knop's salt solution enriched with sucrose.

Haberlandt is regarded as the father of plant tissue culture.

Short history of Plant Tissue Culture

Toward Commercial Micropropagation 1950s Morel & Martin 1952 used Meristem-tip culture for disease elimination. They recovered for the first time, virus- free Dahlia plants

Short history of Plant Tissue Culture

Morel 1960 Disease eradication

Wimber 1963 & in vitro production of orchids

Short history of Plant Tissue Culture

Commercialization of Micropropagation Murashige and Skoog(1962) Developed a universally used high salt medium containing mineral salts, vitamins, an energy source and growth hormone (MS medium)

Murashige(1974) Broad commercial application

What is required?

Tissue culture has several critical requirements:Appropriate tissue.A suitable growth medium which can be liquid or semisolid.Aseptic (sterile) conditions, as microorganisms grow much more quickly than plant and animal tissue and can over run a culture.

Tissue culture environment

General Laboratory and media preparation room

Transfer area

Culturing facilities/Growth rooms

Washing and storage rooms

Greenhouses and containment room

General laboratory & media preparation room

Transfer area (GRC IITA IBADAN)

Design of a Laminar flow hood

A section of a culture room

Timer switch to control photoperiod

Fluorescent tube placed above shelf for lighting

A level of shelf containing in vitro cultures

Hardening process( Acclimatization)

Micropropagation

“… the art and science of multiplying plants in vitro

It implies

- regeneration

- multiplication

- uniformity ??

Starting material for Micropropagation (Explant)

Propagation from

plant cells, tissues or

organs under aseptic

conditions in synthetic

medium in vitro.

Many different explants can be used for micropropagation, but axillary buds and meristems are most commonly used

Tip bud

Leaf

Axillary bud

Internode

Root

Stages of Micropropagation

Stage I - Selection & preparation of the mother plant – sterilization of the plant tissue takes place Initiation of culture – explant placed into growth mediaStage II – Multiplication – explant transferred to shoot media; shoots can be constantly divided.Stage III - Rooting– explant transferred to root mediaStage IV - Transfer to soil– explant returned to soil; hardened off

Stages of micropropagation

STAGE IV: Transfer to Natural EnvironmentSTAGE III: Pretransplant (rooting)

STAGE II: Shoot ProductionSTAGE I - Sterilization

Growth mediumThe growth medium used depends on the plant species being grown. The medium contains the following components:

all of the minerals and vitamins needed for plant growth and differentiation;

a carbon/energy source such as sugar as the explant cannot usually photosynthesize;

various growth regulators to encourage cell enlargement, division and differentiation;

agar which is used to solidify the medium.

Plant growth regulatorsTwo major hormones affect Plant Differentiation:– Auxins: Stimulates Root Development & Cytokinin: Stimulates Shoot Development

Generally, the ratio of these two hormones can determine plant development:

– ↑ Auxin ↓Cytokinin = Root Development

– ↑ Cytokinin ↓Auxin = Shoot Development

– Auxin = Cytokinin = Callus

Hormonal balanceAuxin Cytokinin High Low

Root formation on cuttingsEmbryogenesis

Adventitious root formation in callusCallus initiation

Adventitious shoot formationAxillary shoot growth

Low High

Methods of micropropagation

Clonal propagation involves the multiplication of genetically identical lines by asexual reproduction.

Micropropagation involves the use of bud culture, meristem and shoot-tip culture techniques to introduce plants in vitro by induction to form adventitious buds, shoots and entire plants.

Methods of micropropagation cont’d

Bud culture is the culture of plant buds which contains active meristems.

Meristem culture is the culture of apical meristems which are capable of active cell division and differentiation into specialized and permanent tissue such as shoots and roots

Methods of micropropagation cont’d

Single node culture is done on a hormone free medium.

Axillary bud culture is done using excised shoot tips cultured on medium amended with high cytokinin concentration 6 Benzyl amino purine(BAP)/Benzyl adenine(BA)

Bud culture by single nodes

Surface sterilization in NaOCL

Rinsing with SDW Transfer into sterile container for 2nd

sterilization

Steps in single node culture

1 2 3

45

Methods of micropropagation cont’d

Micro cuttings of yam during and after subculture

3 4

In vitro plantlet regeneration via meristem

& shoot tip culture

Meristem cultureA meristem is an undifferentiated but determined tissue, the cells of which are capable of active cell division and differentiation into specialized and permanent tissues such as shoots and roots.

It has the capacity of producing a complete plant in vivo and in vitro (Totipotent)

Shoot apical meristem (Anderson,1999)

Meristem excison using stereomicroscopes

Stages in meristem excisionDumet et al., 2008

Shoot tip culture

Clonal in vitro propagation by repeated enhanced formation of axillary shoots from shoot-tips cultured on media supplemented with plant growth regulators, usually cytokinins.

The size of the shoot tip is between 0.5 to 5mm and consists of primordia leaves.

Stages in acclimatization of Cassava

1 2 3

4 5

Acclimatization of Yam using constructed humidity chamber

Acclimatization process using polyethylene bags

Problems in Micropropagation

There is problem of genetic variability due to Somaclonal variation in some cultures.

Contamination is a major problem which could cause high losses within a short period.

It requires expensive equipments and well trained manpower.

Vitrification may occur which reduces the rate of growth multiplication of the plant and eventually causes death.

Benefits of micropropagation

Rapid multiplication of superior clones can be carried out through out the year, irrespective of seasonal variations.

Multiplication of disease free plants e.g. virus free plants of sweet potato (Ipomea batatas), cassava (Manihot esculenta) e.t.c.

It is a cost effective process as it requires minimum growing space.

Growth Media– Minerals, Growth factors, Carbon source, HormonesEnvironmental Factors– Light, Temperature, Photoperiod.Explant Source– Usually, the younger, less differentiated the explant, the better for tissue cultureGenetics– Different species show differences in amenability to tissue culture. In many cases, different genotypes within a species will have variable responses to tissue culture.

Factors affecting Tissue Culture

Applications of plant tissue culture

Agriculture

Anther & pollen culture-Production of haploid plants.

Applications of plant tissue culture

Production of virus-free plants for safe germplasm transfer.

Screening of in vitro lines for stress and disease resistance.

Somaclonal Variations has been used in plant breeding programmes where the genetic variations with desired or improved characters are introduced into the plants.

Somaclonal variationSomaclonal variation is a general phenomenon of all plant regeneration systems that involve a callus phase.

There are two general types of Somaclonal Variation:

Heritable, genetic changes (alter the DNA).

Stable, but non-heritable changes (alter gene expression).

Germplasm conservationAn extension of micropropagation techniques through two methods:

Slow growth techniques e.g.: ↓ Temp., ↓ Light, media supplements ( growth retardants).Medium-term storage (1 to 4 years)

Cryopreservation.Ultra low temperatures in liquid nitrogen at -196oC.

Stops cell division & metabolic processes

Very long-term (indefinite?)

Clonal germplasm flow from medium-term storage to long term storage

D.Dumet, 2009. IITA

Cryopreservation

Micropropagation Unit, Royal Botanic Gardens, Kew UK.Cryopreservation is a valuable technology for the cost-effective,sustained long-term conservation of plant genetic material.

Cryopreservation Requirements

Preculturing– Usually a rapid growth rate to create cells with small vacuoles and low water content.

Cryoprotection– Glycerol, DMSO, PEG, etc…, to protect against ice damage and alter the form of ice crystals.

Freezing– The most critical phase; one of two methods is used:

Slow freezing which allows for cytoplasmic dehydration.

Quick freezing which 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 (don’t forget you have to get a plant)

– Thawed cells must be washed of cryoprotectants and nursed back to normal growth.

– Avoid callus production to maintain genetic stability.

Applications of plant tissue culture

Horticulture and Forestry:Micropropagation method is used for rapid multiplication of ornamental plants as well as important trees yielding high fuel, pulp, fruits or oil at a large scale.

Applications of plant tissue culture

Today's high yielding oil palms on plantations in Malaysia were generated in the 1960s by tissue culture methods.

These palms produce 30% more oil than normally cultured palms.

Improvement of economically important forest trees is being done through genetic transformation and rapid Micropropagation .e.g in vitro regeneration and genetic transformation of conifers.

Applications of plant tissue culture

Industries:Plant cell culture is used for biotransformation (modification of functional groups of organic compounds by living cells). Food and agricultural biotechnologists are involved in using tools of molecular biology to enhance the quality and quantity of foods and economic crops. For example, Golden Rice was genetically enhanced with added beta carotene, which is a precursor to Vitamin A in the human body. Plant cells can be cultured in fermenters for the industrial production of secondaryMetabolites using cell culture.

PLANT SPECIES AND SECONDARY METABOLITES OBTAINED FROM THEM USING TISSUE CULTURE

TECHNIQUESProduct Plant source Uses

Artemisin Artemisia spp Antimalarial

Capsaicin Capsicum annum

Cures Rheumatic pain

Codeine Papaver spp. Analgesic

Camptothecin Campatotheca accuminata

Anticancer

Quinine Cinchona officinalis

Antimalarial

At Bells University

Plant tissue culture practicalsCapsicum annum (Red Pepper) regenerated via seed culture

Apple regenerated via embryo culture

Apple subcultured into test tubes using micropropagation

At Bells University

Yam micropropagation Cassava micropropagation

Possible areas for research

Somatic embryogenesis of white Guinea yam(Dioscorea rotundata).

In vitro regeneration of Bambara groundnut (Vigna subterranea L.Verdc) via mersitem culture.

In vitro micropropagation of medicinal plants.

ConclusionThe ability to regenerate entire plants from cells or tissues has been invaluable to plant biotechnology due to the totipotent nature of plant cells.From the sole objective of demonstrating the totipotency of differentiated plant cells, the technique now finds application in both basic and applied researches in number of fields of enquiry. Plant tissue culture has generated intense interest among molecular biologists, plant breeders and commercial horticulturists.This technique should therefore be encouraged and supported by African countries as it could aid in improving food security in Africa.

ReferencesD.Dumet,A.Adeyemi,O.B.Ojuederie (2008). Yam invitro genebanking. Genebank manual. http://www.iita.org/genebank/manualD.Dumet,A.Adeyemi,O.B.Ojuederie (2008).Cassava in vitro processing and the genebanking.Genebank manual. http://www.iita.org/genebank/manualHORT689/AGRO689 Biotechniques in Plant BreedingH.S Chawla .2002 Introduction to Plant Biotechnology 2nd edition. Oxford & IBH Publishing C./ Pvt. Ltd New Delhi IndiaMurashige T. and Skoog F. (1962). A revised medium for rapid growth and bioassay withTobacco tissue culture. Physiologia plantarum 15: 473-497.