Plant Tissue Culture Application

Development of superior cultivars

Germplasm storage Somaclonal variation

Embryo rescue Ovule and ovary cultures

Anther and pollen cultures Callus and protoplast culture

Protoplasmic fusion In vitro screening

Multiplication

Tissue Culture ApplicationsMicropropagation

Germplasm preservationSomaclonal variation

Haploid & dihaploid productionIn vitro hybridization – protoplast

fusion

Micropropagation

Micropropagation advantages From one to many propagules rapidly Multiplication in controlled laboratorium

conditions Continuous propagation year round Potential for disease-free propagules Inexpensive per plant once established Precise crop production scheduling Reduce stock plant space

Micropropagation disadvantagesSpecialized equipment/facilities

requiredMore technical expertise required

Protocols not optimized for all species

Plants produced may not fit industry standards

Relatively expensive to set up

Micropropagation applicationsRapid increase of stock of new

varietiesElimination of diseasesCloning of plant types not easily

propagated by conventional methods (few offshoots/ sprouts/ seeds; date palms, ferns)

Propagules have enhanced growth features (multibranched character)

Methods of micropropagationAxillary branching

Adventitious shoot formation (organogenesis)

Somatic embryogenesis

• >95% of all micropropagation

• Genetically stable• Simple and

straightforward

• Efficient but prone to genetic instability

• Little used. Potentially phenomenally efficient

Axillary shoot proliferationGrowth of axillary buds stimulated by cytokinin treatment; shoots arise mostly from pre-existing meristems

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

Shoots produced are either rooted first in vitro or rooted and acclimatized ex vitro

Steps of micropropagation (axillary branching and adventitious shoot

formation)• Stage 0 – Selection & preparation of the mother

plantSterilization of the plant tissue

• Stage I  - Initiation of cultureExplants placed into growth media

• Stage II - MultiplicationExplants transferred to shoot media; shoots can be

constantly divided• Stage III - Rooting

Explants transferred to root media• Stage IV - Transfer to soil

Explants returned to soil; hardened off

Procedures for cleaning virus infected clones and subsequent generation of nuclear seed potatoes for

distribution

Clean Stock Program Used for Commercial Potato

Seed Potato ProductionA

C D

B

Shoots (A) from virus-free merstems multiplied in vitro (B) are transferred into soil medium and grown in a screened greenhouse (C, D) to ward off insect vectors

Ways to eliminate viruses Heat treatment.

Plants grow faster than viruses at high temperatures.

Meristemming. Viruses are transported from cell to cell through plasmodesmata and through the vascular tissue. Apical meristem often free of viruses. Trade off between infection and survival.

Not all cells in the plant are infected.Adventitious shoots formed from single cells can give virus-free shoots.

Elimination of virusesPlant from the field

Pre-growth in the greenhouse

‘Virus-free’ Plants

Heat treatment35oC / months

Activegrowth

Meristem culture

Micropropagation cycle

Virus testing

AdventitiousShoot formation

Somatic EmbryogenesisExplant → Callus Embryogenic → Maturation →

Germination

1.Callus induction2. Embryogenic callus

development3.Maturation

4.Germination

Induction• Auxins required for induction

–Proembryogenic masses form–2,4-D most used–NAA, dicamba also used

DevelopmentAuxin must be removed for embryo

developmentContinued use of auxin inhibits embryogenesisStages are similar to those of zygotic

embryogenesis– Globular– Heart– Torpedo– Cotyledonary– Germination (conversion)

Maturation• Require complete maturation with apical

meristem, radicle, and cotyledons• Often obtain repetitive embryony• Storage protein production necessary• Often require ABA for complete

maturation• ABA often required for normal embryo

morphology – Fasciation– Precocious germination

Germination• May only obtain 3-5% germination• Sucrose (10%), mannitol (4%) may be

required• Drying (desiccation)

– ABA levels decrease– Woody plants– Final moisture content 10-40%

• Chilling– Decreases ABA levels– Woody plants

Peanut somatic embryogenesis

In situ : Conservation in ‘normal’ habitat–rain forests, gardens, farms

Ex Situ : –Field collection, botanical gardens –Seed collections –In vitro collection: Extension of micropropagation techniques

•Normal growth (short term storage)•Slow growth (medium term storage)•Cryopreservation (long term storage)

DNA Banks

Plant germplasm preservation

In vitro CollectionPotential advantages of in vitro methods: little space needsplants are free of pests,

pathogens and viruses (and will remain so)

no transfer labor (under storage conditions)

stored cultures can be used as nuclear stock for vegetative preservation

international shipping restrictions are lessened

1. no soil2. pest-free plants

In vitro Collection• Basic goals of an in vitro storage

system– to maintain genetic stability– to keep in indefinite storage without loss

of viability– must be economical

• Two/three types of systems:– Normal growth– Slow growth– Cryopreservation

Normal Growth1.It can be done either on semi solid

media or in liquid media2.It is similar to multiplication stage in

micro-propagation3.It must be frequently sub-cultured4.When axillary buds are used as explants,

it is considered as genetically stabile

Slow growthIt can store at least 1 semester and maximum 6 years without sub-culturing

Ways to achieve slow growth: Addition of inhibitors or retardants Increasing osmotic potential of the

media Manipulating storage temperature and

light (cold storage (1-9° C)) Reducing light intensity Mineral oil overlay (callus) Reduced oxygen tension

Plant Growth Retardants

any chemicals that slow cell division and elongation in shoot tissues

Cause plants to be shorter and more compactInterrupts cell division, stem elongation, and seed head formationRoots continue to growMay reduce the natural Gibberellic acidMay produce more ethylene

Cold storagestorage at non-freezing temps, from 1-9°

C dependent on species.storage of shoot cultures (stage I or II)

• works well for strawberries, potatoes, grapes, prob. many more spp.

• transferred (to fresh medium) every 6 month or on a yearly basis

Advantages: simple, high rates of survival, useful for micro-propagation (especially in

periods of low demand)

•It may not be suitable for tropical, subtropical species because of susceptibility of these to chill injury

•It is an alternative with coffee – shoot cultures transferred to a medium with reduced nutrients and lacking sucrose

•It requires refrigeration, which is more expensive than storage in cryopreservation

Disadvantages

In vitro storage of 10 C

Conservation of plant germplasm • Vegetatively propagated species (root and tubers,

ornamental, fruit trees)• Recalcitrant seed species (Howea, coconut, coffee)

Conservation of tissue with specific characteristics• Medicinal and alcohol producing cell lines• Genetically transformed tissues• Transformation/Mutagenesis competent tissues (ECSs)

Eradication of viruses (Banana, Plum)Conservation of plant pathogens (fungi, nematodes)

CryopreservationStorage of living tissues at ultra-low temperatures

(-196°C)

Cryopreservation Steps Selection

Excision of plant tissues or organs Culture of source material Select healthy cultures Apply cryo-protectants Pre-growth treatments

Cooling/freezing Storage

Warming & thawing Recovery growth Viability testing Post-thawing

Cryopreservation Requirements• Preculturing

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

• Cryoprotection– Cryoprotectant (Glycerol, DMSO/dimetil

sulfoksida, 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 cryo-protectants and nursed back to normal growth

– Avoid callus production to maintain genetic stability

Somaclonal Variation Variation found in somatic cells dividing mitotically in

culture A general phenomenon of all plant regeneration systems

that involve a callus phase Variation in trait(s) generated by use of a tissue-culture

cycle Genetic variations in plants that have been produced by

plant tissue culture and can be detected as genetic or phenotypic traits

Two general types of Somaclonal Variation:– Heritable, genetic changes (alter the DNA)– Stable, but non-heritable changes (alter gene expression,epigenetic)

Genetic (Heritable Variations)•Pre-existing variations in the somatic cells of explant

•Caused by mutations and other DNA changes•Occur at high frequency

•Variations generated during tissue culture•Caused by temporary phenotypic changes•Occur at low frequency

Epigenetic (Non-heritable Variations)

Causes of Somaclonal Variations

Physiological Cause

Genetic Cause

Biochemical Cause

1. Change in chromosome number2. Change in chromosome structure3. Gene Mutation4. Extrachomosomal gene mutation5. Transposable element activation6.6. DNA sequenceDNA sequence

Genetic Cause

Change in DNA Change in DNA Detection of altered fragment size by Detection of altered fragment size by using Restriction enzymeusing Restriction enzyme

Change in ProteinChange in Protein Loss or gain in protein bandLoss or gain in protein band Alteration in level of specific proteinAlteration in level of specific protein

Methylation of DNAMethylation of DNA Methylation inactivates transcription Methylation inactivates transcription processprocess

DNA sequence

Advantages of Somaclonal Variations

• Help in crop improvement• Creation of additional genetic varitaions• Increased and improved production of

secondary metabolites• Selection of plants resistant to various

toxins, herbicides, high salt concentration and mineral toxicity

• Suitable for breeding of perrenial species

Disadvantages of Somaclonal Variations

• A serious disadvantage occurs in operations which require clonal uniformity, as in the horticulture and forestry industries where tissue culture is employed for rapid propagation of elite genotypes

• Sometime leads to undesirable results• Selected variants are random and genetically

unstable• Require extensive and extended field trials • Not suitable for complex agronomic traits like

yield and quality• May develop variants with pleiotropic effects

which are not true.

Somaclonal Breeding Procedures• Use plant cultures as starting material

– Idea is to target single cells in multi-cellular culture– Usually suspension culture, but callus culture can work

(want as much contact with selective agent as possible)– Optional: apply physical or chemical mutagen

• Apply selection pressure to culture– Target (very high kill rate)– Generate screening dosage (lethal dosage is dependent

upon the expected number survive cells• Regenerate whole plants from surviving

cells - Direct organogenesis or embryogenesis

Requirements for Somaclonal Breeding• Effective screening procedure

– Most mutations are deleterious• With fruit fly, the ratio is ~800:1 deleterious to

beneficial– Most mutations are recessive

• Must screen M2 or later generations• Consider using heterozygous plants?

– But some say you should use homozygous plants to be sure effect is mutation and not natural variation

• Haploid plants seem a reasonable alternative if possible

– Very large populations are required to identify desired mutation: • Can you afford to identify marginal traits with

replicates & statistics? Estimate: ~10,000 plants for single gene mutant

• Clear Objective

Embryo Culture Uses•Rescuing interspecific and intergeneric

hybrids– wide hybrids often suffer from early spontaneous abortion– cause is embryo-endosperm failure– Gossypium, Brassica, Linum, Lilium•Production of monoploids– useful for obtaining "haploids" of barley, wheat, other

cereals– the barley system uses Hordeum bulbosum as a pollen

parent

Embryo Culture of Citrus

Coconut embryo culture

Bulbosum MethodHordeum vulgareBarley

2n = 2X = 14

Hordeum bulbosum

Wild relative2n = 2X = 14

Haploid Barley2n = X = 7

H. Bulbosum chromosomes

eliminated

X

Embryo Rescue↓

• This was once more efficient than microspore culture in creating haploid barley

• Now, with an improved culture media (sucrose replaced by maltose), microspore culture is much

more efficient (~2000 plants per 100 anthers)

Bulbosum techniqueHordeum vulgare is the seed parentzygote develops into an embryo with elimination

of Hordeum bulbosum chromosomeseventually, only HV chromosomes are leftembryo is "rescued“ to avoid abortion

Excision of the immature embryo: Hand pollination of freshly opened flowers Surface sterilization – EtOH on enclosing

structures Dissection – dissecting under microscope

necessary Plating on solid medium – slanted media are

often used to avoid condensation

Culture Medium–Mineral salts – K, Ca, N most

important–Carbohydrate and osmotic pressure

– Amino acids– Plant growth regulators

Culture Medium–Carbohydrate and osmotic pressure» 2% sucrose works well for mature embryos» 8-12% for immature embryos» transfer to progressively lower levels as embryo grows» alternative to high sucrose – auxin & cyt PGRs–amino acids» reduced N is often helpful» up to 10 amino acids can be added to replace N salts,

incl. glutamine, alanine, arginine, aspartic acid, etc.» requires filter-sterilizing a portion of the medium

– natural plant extracts» coconut milk (liquid endosperm of coconut)» enhanced growth attributed to undefined hormonal

factors and/or organic compounds» others – extracts of dates, bananas, milk, tomato juice– PGRs» globular embryos – require low conc. of auxin and

cytokinin» heart-stage and later – usually none required» GA and ABA regulate "precocious germination“» GA promotes, ABA suppresses

Culture Medium

“Wide” crossing of wheat and rye requires embryo rescue and chemical treatment to

double the number of chromosomes.

Triticale

Haploid Plant Production Embryo rescue of

interspecific crosses– Creation of alloploids

Anther culture/Microspore culture– Culturing of Anthers or

Pollen grains (microspores)

– Derive a mature plant from a single microspore

Ovule culture– Culturing of unfertilized

ovules (macrospores)

Initiation from Stamens and Pistils

Embryogenic callus

Callus formation from connective tissue

Callus formation from filament tip

Embryo developmentEmbryo germination

Stamen explant

Poliploidization

Specific Examples of DH uses• Evaluate fixed progeny from an F1

– Can evaluate for recessive & quantitative traits– Requires very large dihaploid population, since no prior

selection– May be effective if you can screen some qualitative traits

early• For creating permanent F2 family for molecular

marker development• For fixing inbred lines (novel use?)

– Create a few dihaploid plants from a new inbred prior to going to Foundation Seed (allows you to uncover unseen off-types)

• For eliminating inbreeding depression (theoretical)– If you can select against deleterious genes in culture, and

screen very large populations, you may be able to eliminate or reduce inbreeding depression

– e.g.: inbreeding depression has been reduced to manageable level in maize through about 50+ years of breeding; this may reduce that time to a few years for a crop like onion or alfalfa

Somatic HybridizationDevelopment of hybrid plants through the

fusion of somatic protoplasts of two different plant species/varieties

Somatic hybridization technique

1. isolation of protoplast1. isolation of protoplast

2. Fusion of the protoplasts of desired species/varieties2. Fusion of the protoplasts of desired species/varieties

3. Identification and Selection of somatic hybrid cells3. Identification and Selection of somatic hybrid cells

4. Culture of the hybrid cells4. Culture of the hybrid cells

5. Regeneration of hybrid plants 5. Regeneration of hybrid plants

Isolation of Protoplast (Separartion of protoplasts from plant tissue))

1. Mechanical Method 2. Enzymatic Method

Mechanical Method

Plant Tissue

Collection of protoplasm

Cells Plasmolysis

Microscope Observation of cells

Cutting cell wall with knife Release of protoplasm

Mechanical Method

Used for vacuolated cells like onion bulb scale, radish and beet root tissues

Low yield of protoplastLaborious and tedious processLow protoplast viability

Enzymatic MethodLeaf sterlization, removal of epidermis

Plasmolysed cells

Plasmolysed cells

Pectinase +cellulase Pectinase

Protoplasm released Release of isolated cells

cellulase

Protoplasm released

Isolated Protoplasm

Enzymatic Method

Used for variety of tissues and organs including leaves, petioles, fruits, roots, coleoptiles, hypocotyls, stem, shoot apices, embryo microspores

Mesophyll tissue - most suitable source High yield of protoplast Easy to perform More protoplast viability

Protoplast FusionProtoplast Fusion(Fusion of protoplasts of two different genomes(Fusion of protoplasts of two different genomes))

1. Spontaneous Fusion 2. Induced Fusion

Intraspecific Intergeneric ElectrofusionMechanical Fusion

Chemofusion

Uses for Protoplast FusionCombine two complete genomes

– Another way to create allopolyploids In vitro fertilizationPartial genome transfer

– Exchange single or few traits between species– May or may not require ionizing radiation

Genetic engineering– Micro-injection, electroporation, Agrobacterium

Transfer of organelles– Unique to protoplast fusion– The transfer of mitochondria and/or chloroplasts

between species

Spontaneous Fusion• Protoplast fuse spontaneously

during isolation process mainly due to physical contact

• Intraspecific produce homokaryones• Intergeneric have no importance

Induced Fusion

• Types of fusogens• PEG• NaNo3

• Ca 2+ ions• Polyvinyl alcohol

Chemofusion- fusion induced by chemicals

Induced Fusion• Mechanical Fusion- Physical fusion of

protoplasts under microscope by using micromanipulator and perfusion micropipette

• Electrofusion- Fusion induced by electrical stimulation

• Fusion of protoplasts is induced by the application of high strength electric field (100kv m-1) for few microsecond

Possible Result of Fusion of Two Genetically Different Protoplasts

= chloroplast

= mitochondria

= nucleusFusion

heterokaryon

cybrid cybridhybrid hybrid

Identifying Desired Fusions• Complementation selection

– Can be done if each parent has a different selectable marker (e.g. antibiotic or herbicide resistance), then the fusion product should have both markers

• Fluorescence-activated cell sorters– First label cells with different fluorescent markers;

fusion product should have both markers• Mechanical isolation

– Tedious, but often works when you start with different cell types

• Mass culture– Basically, no selection; just regenerate everything

and then screen for desired traits

Advantages of somatic hybridization

• Production of novel interspecific and intergenic hybrid– Pomato (Hybrid of potato and tomato)

• Production of fertile diploids and polypoids from sexually sterile haploids, triploids and aneuploids

• Transfer gene for disease resistance, abiotic stress resistance, herbicide resistance and many other quality characters

• Production of heterozygous lines in the single species which cannot be propagated by vegetative means

• Studies on the fate of plasma genes• Production of unique hybrids of nucleus and

cytoplasm

Problem and Limitation of Somatic Hybridization

1. Application of protoplast technology requires efficient plant regeneration system.

2. The lack of an efficient selection method for fused product is sometimes a major problem.

3. The end-product after somatic hybridization is often unbalanced.

4. Development of chimaeric calluses in place of hybrids.5. Somatic hybridization of two diploids leads to the formation of

an amphiploids which is generally unfavorable.6. Regeneration products after somatic hybridization are often

variable.7. It is never certain that a particular characteristic will be

expressed.8. Genetic stability.9. Sexual reproduction of somatic hybrids.10.Inter generic recombination.

TYPICAL SUSPENSION PROTOPLAST + LEAF PROTOPLAST PEG-INDUCED FUSION

NEW SOMATIC HYBRID PLANT

True in vitro fertilization

Using single egg and sperm cells and fusing them electrically

Fusion products were cultured individually in 'Millicell' inserts in a layer of feeder cells

The resulting embryo was cultured to produce a fertile plant

A procedure that involves retrieval of eggs and sperm from the male and

female and placing them together in a laboratory dish to facilitate

fertilization

Requirements for plant genetic transformation

• Trait that is encoded by a single gene• A means of driving expression of the gene

in plant cells (Promoters and terminators)

• Means of putting the gene into a cell (Vector)

• A means of selecting for transformants• Means of getting a whole plant back from

the single transformed cell (Regeneration)