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

Features of Micropropagation• Clonal reproduction

– Way of maintaining heterozygozity• Multiplication stage can be recycled many

times to produce an unlimited number of clones– Routinely used commercially for many ornamental

species, some vegetatively propagated crops• Easy to manipulate production cycles

– Not limited by field seasons/environmental influences

• Disease-free plants can be produced– Has been used to eliminate viruses from donor

plants

Microcutting propagation• It involves the production of shoots from

pre-existing meristems only.• Requires breaking apical dominance

• This is a specialized form of organogenesis

Steps of Micropropagation• Stage 0 – Selection & preparation of the mother

plant– sterilization of the plant tissue takes place

• Stage I  - Initiation of culture– explant placed into growth media

• Stage II - Multiplication– explant transferred to shoot media; shoots

can be constantly divided• Stage III - Rooting

– explant transferred to root media• Stage IV - Transfer to soil

– explant returned to soil; hardened off

Conventional Micropropagation

Duration: 6 years 2 years

Labor: Dig & replant every 2 years; Subculture every 4 weeks;

unskilled (Inexpensive) skilled (more expensive)

Space: More, but less expensive (field) Less, but more expensive (laboratory)

Required to prevent viral Screening, fumigation, spraying Noneinfection:

COMPARISON OF CONVENTIONAL & MICROPROPAGATION OF VIRUS

INDEXED REGISTERED RED RASPBERRIES

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

Indirect 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

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

Use : Recalcitrant seeds Vegetatively

propagated Large seeds

In vitro Collection

Concern: SecurityAvailabilitycost

Use of immature zygotic embryos (not for vegetatively propagated species)

Addition of inhibitors or retardants Manipulating storage temperature and

light Mineral oil overlay

Reduced oxygen tension Defoliation of shoots

Ways to achieve slow growth

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

Some mechanisms: Karyotipic alteration Sequence variation

Variation in DNA Methylation

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

expression, epigenetic)

Epigeneticthe study of gene regulation that does not involve making changes to the SEQUENCE of the DNA,

but rather to the actual BASES within the nucleotides and to the HISTONES

The three main mechanisms for regulation are: CpG island methylation (…meCGmeCGmeCGmeCGmeCGmeCGmeCGmeCG…) acetylation and methylation of histone H3 the production of antisense RNA

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, you want very few cells to survive, so long as selection is effective

• Regenerate whole plants from surviving cells

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

– Can’t expect to just plant things out and see what happens; relates to having an effective screen

– This may be why so many early experiments failed

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

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 techniqueH. vulgare is the seed parentzygote develops into an embryo with elimination

of HB 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)

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 Method

Leaf 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)