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.