MICROPROPAGATION

IntroductionIntroduction In nature plants propagate eitherIn nature plants propagate either SexuallySexually (seeds generation) results heterogeneity (seeds generation) results heterogeneity

OrOr AsexuallyAsexually (vegetative multiplication) produce (vegetative multiplication) produce

genetically identical plants. genetically identical plants.

Multiplication of genetically identical copies of a Multiplication of genetically identical copies of a cultivar by asexual reproduction is called clonal cultivar by asexual reproduction is called clonal propagation.propagation.

Via tissue culture called micropropagation,Via tissue culture called micropropagation,

Basic in vitro propagation ...

What is What is Micropropagation?Micropropagation?

“… the asexual or vegetative propagation (multiplication) of plants in vitro “

Implies - regeneration - multiplication - uniformity ??

MicropropagationMicropropagation

• The art and science of plant The art and science of plant multiplication multiplication in vitroin vitro

• Usually derived from meristems (or Usually derived from meristems (or vegetative buds) without a callus stagevegetative buds) without a callus stage– Tends to reduce or eliminate somaclonal Tends to reduce or eliminate somaclonal

variation, resulting in true clonesvariation, resulting in true clones

• Can be derived from other explant or Can be derived from other explant or callus (but these are often problematic)callus (but these are often problematic)

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

Features of Features of MicropropagationMicropropagation• Clonal reproductionClonal reproduction

– Way of maintaining heterozygozityWay of maintaining heterozygozity

• Multiplication Stage can be recycled many Multiplication Stage can be recycled many times to produce an unlimited number of clonestimes to produce an unlimited number of clones– Routinely used commercially for many ornamental Routinely used commercially for many ornamental

species, some vegetatively propagated cropsspecies, some vegetatively propagated crops

• Easy to manipulate production cyclesEasy to manipulate production cycles– Not limited by field seasons/environmental Not limited by field seasons/environmental

influencesinfluences

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

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 disadvantages

Specialized equipment/facilities required

More technical expertise required

Protocols not optimized for all species

Plants produced may not fit industry standards

Relatively expensive to set up

Micropropagation applications

Rapid increase of stock of new varieties

Elimination of diseases

Cloning of plant types not easily propagated by conventional methods (few offshoots/ sprouts/ seeds; date palms, ferns)

Propagules have enhanced growth features (multibranched character)

Micropropagation Micropropagation (contin)(contin)

Positives and negatives of Positives and negatives of micropropagationmicropropagation positivespositives

rapid multiplication ratesrapid multiplication rates low space requirementlow space requirement

negativesnegatives labor costslabor costs expensive (equipment, facilities, supplies)expensive (equipment, facilities, supplies) loss by contaminationloss by contamination danger of variationdanger of variation

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

plantplant– sterilization of the plant tissue takes placesterilization of the plant tissue takes place

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

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

constantly dividedconstantly divided

• Stage III - RootingStage III - Rooting– explant transferred to root mediaexplant transferred to root media

• Stage IV - Transfer to soilStage IV - Transfer to soil– explant returned to soil; hardened offexplant returned to soil; hardened off

STAGES

1. Selection of plant material

2. Establish aseptic culture

3. Multiplication

4. Shoot elongation

5. Root induction / formation

6. Acclimatization

STAGES

1. Selection of plant material

2. Establish aseptic culture

3. Multiplication

4. Shoot elongation

5. Root induction / formation

6. Acclimatization

Starting material formicropropagation

Leaf

Tip bud

Internode

Axillary bud

Root

Selection of plant material ...

Part of plant

Genotype

Physiological condition

Season

Position on plant

Size of explant

Physiological state - of stock plant

Vegetative / Floral

Juvenile / Mature

Dormant / Active

Carbohydrates

Nutrients

Hormones

STAGES

1. Selection of plant material

2. Establish aseptic culture

3. Multiplication

4. Shoot elongation

5. Root induction / formation

6. Acclimatization

Disinfestation

Stock plant preparation

Washing in water

Disinfecting solution

Internal contaminants

Screening

The medium

Minerals

Sugar

Organic ‘growth factors’

Growth regulators

Gelling agent

Other additives

Physical Environment

Temperature

Moisture

Light

STAGES

1. Selection of plant material

2. Establish aseptic culture

3. Multiplication

4. Shoot elongation

5. Root induction / formation

6. Acclimatization

Origins of new shoots ...

Terminal extension

Lateral / Axillary buds

Adventitious (de novo, re-differentiation)

Callus differentiation

Role of growth regulators ...

Cell division

Differentiation

Cell expansion

Apical dominance

auxins

abscisic acid

cytokinins

gibberelic acid

ethylene

STAGES

1. Selection of plant material

2. Establish aseptic culture

3. Multiplication

4. Shoot elongation

5. Root induction / formation

6. Acclimatization

Shoot elongation ...

Basal ‘hormone free’ medium

Gibberellins

Carry-over of hormones

STAGES

1. Selection of plant material

2. Establish aseptic culture

3. Multiplication

4. Elongation

5. Root induction / formation

6. Acclimatization

Root initiation ...

Auxins

Co-factors

C : N ratio

Light / darkness

Initiation vs growth

Juvenility / rejuvenation

Genotype

STAGES

1. Selection of plant material

2. Establish aseptic culture

3. Multiplication

4. Elongation

5. Root induction / formation

6. Acclimatization

- survival of the new plant when removed

from the in vitro environment

Acclimatization (hardening)

Micropropagation of Micropropagation of almost all the fruit crops almost all the fruit crops and vegetables is possibleand vegetables is possible Some examples: dwarfing sweet cherry, Some examples: dwarfing sweet cherry,

Shade trees, Ornamental shrubs, Shade trees, Ornamental shrubs, Roses, Clematis, Lilacs, Saskatoon Roses, Clematis, Lilacs, Saskatoon berries, Nutraceutical Plants, berries, Nutraceutical Plants, Rhododendron, Azalea, mustard, corn, Rhododendron, Azalea, mustard, corn, soybeans, wheat, rice, cotton, tomato, soybeans, wheat, rice, cotton, tomato, potato, citrus, turf, legumespotato, citrus, turf, legumes

Advantages of Advantages of MicropropagationMicropropagation economical in time and space economical in time and space greater output -can produce millions of greater output -can produce millions of

uniformly flowering and yielding plantsuniformly flowering and yielding plants African Biotechnologies - fruit crops African Biotechnologies - fruit crops

banana and indoor pot flowers- 6 million banana and indoor pot flowers- 6 million pieces per yearpieces per year

disease free disease free elite plants with exceptional elite plants with exceptional

characteristicscharacteristics

Advantages Cont’dAdvantages Cont’d

facilitates safer movements of facilitates safer movements of germplasmgermplasm across nations - across nations - In vitroIn vitro germplasm assures the exchange of germplasm assures the exchange of pest and disease free material pest and disease free material

great for great for vegetatively reproduced crops vegetatively reproduced crops crops which produce few seeds or highly crops which produce few seeds or highly

heterozygous seeds.heterozygous seeds.

Uses of Uses of MicropropagationMicropropagation

Used to create transgenic, first Used to create transgenic, first generation plantsgeneration plants

Used in horticulture to produce orchids, Used in horticulture to produce orchids, African Violets, lilies, and fernsAfrican Violets, lilies, and ferns

Used in nurseries to grow fruit trees, Used in nurseries to grow fruit trees, evergreens, roses, and shade treesevergreens, roses, and shade trees

Benefits of Benefits of MicropropagationMicropropagation

Many genetically identical plants can be Many genetically identical plants can be created from one parent plantcreated from one parent plant

Because plants are clones, the uniformity Because plants are clones, the uniformity assures qualityassures quality

Allows many plants to grow in a small Allows many plants to grow in a small place in a short timeplace in a short time

In some species this method will produce In some species this method will produce healthier plantshealthier plants