Micropropagation “… the art and science of multiplying plants in vitro.”

Plant Propagation by Tissue Culture GJ DeKlerk, EF George, MA Hall (eds) 3rd Edition, 2008 Springer http://www.springerlink.com/content/n5tm30/?p=2d1b86aedb5a437f987c7bd63962bbc8&pi=0

Rapid clonal in vitro propagation of plants: from cells, tissues or organs cultured aseptically on defined media contained in culture vessels maintained under controlled conditions of light and temperature

Toward Commercial Micropropagation 1950s Morel & Martin 1952 Meristem-tip culture for disease elimination

Morel 1960 Disease eradication Wimber 1963 & in vitro production of orchids

Commercialization of Micropropagation 1970s & 1980s Murashige 1974 Broad commercial application

Clone Genetically identical assemblage of individuals propagated entirely by vegetative means from a single plant.

Conventional Propagation Cuttings Budding, grafting Layering

Advantages Conventional Propagation Equipment costs minimal Little experience or technical expertise needed Inexpensive Specialized techniques for growth control (e.g. grafting onto dwarfing rootstocks)

Micropropagation Advantages From one to many propagules rapidly Multiplication in controlled lab conditions Continuous propagation year round Potential for disease-free propagules Inexpensive per plant once established

Micropropagation Advantages Precise crop production scheduling Reduce stock plant space Long-term germplasm storage Production of difficult-to-propagate species

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, nandinas) Propagules have enhanced growth features (multibranched character; Ficus, Syngonium)

Explant Cell, tissue or organ of a plant that is used to start in vitro cultures Many different explants can be used for micropropagation, but axillary buds and meristems are most commonly used

Choice of explant Desirable properties of an explant: Easily sterilizable Juvenile Responsive to culture Importance of stock plants Shoot tips Axillary buds Seeds Hypocotyl (from germinated seed) Leaves

Methods of micropropagation Axillary branching Adventitious shoot formation Somatic embryogenesis >95% of all micropropagation Genetically stable Simple and straightforward Efficient but prone to genetic instability Little used, but potentially phenomenally efficient

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

Shoot Culture Method Overview 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

ADVANTAGES Reliable rates and consistency of shoot multiplication 3 -8 fold multiplication rate per month Pre-existing meristems are least susceptible to genetic changes

mericloning  A propagation method using shoot tips in culture to proliferate multiple buds, which can then be separated, rooted and planted out

Through protocorms, 1,000,000 per year. First commercially used with orchids - conventional propagation rate of 1 per year. Through protocorms, 1,000,000 per year. Corm (Swollen stem) Chop into pieces Maturation

Axillary shoot production Selection of plant material Establish aseptic culture Multiplication Shoot elongation Root induction / formation Acclimatization

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

Stage 1

Disinfestation Stock plant preparation Washing in water Disinfecting solution Internal contaminants Screening

Mother Block: A slowly multiplying indexed and stabilized set of cultures Serve as source of cultures (explants) for Stage II multiplication

Stage I - Sterilization Pre-treatments Transfer plants to a greenhouse to reduce endemic contaminants Force outgrowth of axillary buds Washing removes endemic surface contaminants Antibiotics, fungicides, Admire, others Bacteria and fungi will overgrow the explant on the medium unless they are removed Pre-treatments to clean up the explant Detergents Sterilants and Antibiotics

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

Physical Environment Temperature Moisture Light

Problems Contamination Browning of the explant (phenolics) Dormant buds (woody plants)

STAGE II: Shoot Production • Stage II selection of cytokinin type and concentration determined by: Shoot multiplication rate Length of shoot produced Frequency of genetic variability Cytokinin effects on rooting and survival

Origins of new shoots Terminal extension Lateral / Axillary buds Adventitious (de novo, re-differentiation) Callus differentiation

Problems Vitrification – a glassy appearance to tissue Long acclimation time Callus formation leading to mutations

STAGE II: Shoot Production Subculture shoot clusters at 4 -5 week intervals 3 -8 fold increase in shoot numbers Number of subcultures possible is species/cultivar dependent

STAGE II: Shoot Production

STAGE III: Pretransplant (rooting) Goals: Preparation of Stage II shoots/shoot clusters for transfer to soil (prehardening) Elongation of shoots prior to ex vitro rooting Fulfilling dormancy requirements

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

Root initiation Auxins Charcoal C : N ratio Light / darkness Initiation vs growth Juvenility / rejuvenation Genotype

STAGE III: Pretransplant (rooting)

STAGE IV: Transfer to Natural Environment Ultimate success of shoot culture depends on ability to acclimatize vigorously growing quality plants from in vitro to ex vitro conditions

Stage IV

STAGE IV: Transfer to Natural Environment Acclimatization: Process whereby plants physiologically and anatomically adjust from in vitro to ex vitro cultural and environmental conditions Two reasons micropropagated plants may be difficult to acclimatize ex vitro: Low photosynthetic competence (heterotrophic nutrition) Poor control of water loss