MICROPROPAGATION

Introduction In nature plants propagate either Sexually (seeds generation) results heterogeneity Or Asexually (vegetative multiplication) produce genetically identical plants. Multiplication of genetically identical copies of a cultivar by asexual reproduction is called clonal propagation. Via tissue culture called micropropagation,

Basic in vitro propagation ...

What is Micropropagation? “… the asexual or vegetative propagation (multiplication) of plants in vitro “ Implies - regeneration - multiplication - uniformity ??

Micropropagation The art and science of plant multiplication in vitro Usually derived from meristems (or vegetative buds) without a callus stage Tends to reduce or eliminate somaclonal variation, resulting in true clones Can be derived from other explant or callus (but these are often problematic) Basic definition of micropropagation: The art & science of plant multiplication in vitro; an important aspect is that usually meristematic tissue is used as the explant, and a callus stage is avoided. If you avoid the callus stage, you can avoid the consequences often associated with callus culture: somaclonal variation, and true clones are a result; micropropagation can be done with other explants and even callus tissue, but other explants are usually more difficult to increase and as mentioned, a callus stage often has problems.

Methods of micropropagation Axillary 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 proliferation Growth 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 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

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 (contin) Positives and negatives of micropropagation positives rapid multiplication rates low space requirement negatives labor costs expensive (equipment, facilities, supplies) loss by contamination danger of variation

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

STAGES 2. Establish aseptic culture 3. Multiplication 1. Selection of plant material 2. Establish aseptic culture 3. Multiplication 4. Shoot elongation 5. Root induction / formation 6. Acclimatization

2. Establish aseptic culture 3. Multiplication 4. Shoot elongation STAGES 1. Selection of plant material 2. Establish aseptic culture 3. Multiplication 4. Shoot elongation 5. Root induction / formation 6. Acclimatization

Starting material for micropropagation Tip bud Starting material for micropropagation Leaf Axillary bud Internode 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

2. Establish aseptic culture 3. Multiplication 4. Shoot elongation 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

Organic ‘growth factors’ Growth regulators Gelling agent The medium Minerals Sugar Organic ‘growth factors’ Growth regulators Gelling agent Other additives

Physical Environment Temperature Moisture Light

2. Establish aseptic culture 3. Multiplication 4. Shoot elongation STAGES 1. Selection of plant material 2. Establish aseptic culture 3. Multiplication 4. Shoot elongation 5. Root induction / formation 6. Acclimatization

Lateral / Axillary buds Adventitious (de novo, re-differentiation) 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

2. Establish aseptic culture 3. Multiplication 4. Shoot elongation STAGES 1. Selection of plant material 2. Establish aseptic culture 3. Multiplication 4. Shoot elongation 5. Root induction / formation 6. Acclimatization

Carry-over of hormones Shoot elongation ... Basal ‘hormone free’ medium Gibberellins Carry-over of hormones

2. Establish aseptic culture 3. Multiplication 4. Elongation STAGES 1. Selection of plant material 2. Establish aseptic culture 3. Multiplication 4. Elongation 5. Root induction / formation 6. Acclimatization

Juvenility / rejuvenation Genotype Root initiation ... Auxins Co-factors C : N ratio Light / darkness Initiation vs growth Juvenility / rejuvenation Genotype

2. Establish aseptic culture 3. Multiplication 4. Elongation 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) - survival of the new plant when removed from the in vitro environment

Micropropagation of almost all the fruit crops and vegetables is possible Some examples: dwarfing sweet cherry, Shade trees, Ornamental shrubs, Roses, Clematis, Lilacs, Saskatoon berries, Nutraceutical Plants, Rhododendron, Azalea, mustard, corn, soybeans, wheat, rice, cotton, tomato, potato, citrus, turf, legumes

Advantages of Micropropagation economical in time and space greater output -can produce millions of uniformly flowering and yielding plants African Biotechnologies - fruit crops banana and indoor pot flowers- 6 million pieces per year disease free elite plants with exceptional characteristics

Advantages Cont’d facilitates safer movements of germplasm across nations - In vitro germplasm assures the exchange of pest and disease free material great for vegetatively reproduced crops crops which produce few seeds or highly heterozygous seeds.

Uses of Micropropagation Used to create transgenic, first generation plants Used in horticulture to produce orchids, African Violets, lilies, and ferns Used in nurseries to grow fruit trees, evergreens, roses, and shade trees

Benefits of Micropropagation Many genetically identical plants can be created from one parent plant Because plants are clones, the uniformity assures quality Allows many plants to grow in a small place in a short time In some species this method will produce healthier plants

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