MICROPROPAGATION

Plant Tissue Culture The culture of plant seeds, organs, tissues, cells, or protoplasts on nutrient media under sterile conditions.

Basis for Plant Tissue Culture Two Hormones Affect Plant Differentiation: Auxin: Stimulates Root Development Cytokinin: Stimulates Shoot Development Generally, the ratio of these two hormones can determine plant development:  Auxin ↓Cytokinin = Root Development  Cytokinin ↓Auxin = Shoot Development Auxin = Cytokinin = Callus Development

Breeding Applications of Tissue Culture Micropropagation Germplasm Preservation Somaclonal variation Embryo Culture Haploid Production In vitro Hybridization- Protoplast fusion

Micropropagation In vitro Clonal Propagation. Micropropagation is the practice of rapidly multiplying stock plant material to produce a large number of progeny plants, using modern plant tissue culture methods.

Clone Clone is a plant population derived from a single individual by asexual reproduction. Clonal Propagation is the multiplication of genetically identical individuals by asexual reproduction.

Features of Micropropagation Clonal reproduction Multiplication stage can be recycled many times to produce an unlimited number of clones Easy to manipulate production cycles Disease-free plants can be produced

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

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

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

Selection of plant material Part of plant Genotype Physiological condition Season Position on plant Size of explant

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

Steps of Micropropagation Stage I –Establishment Selection of the explant plant Sterilization of the plant tissue takes place Establishment to growth medium Stage II  - Proliferation Transfer to proliferation media Shoots can be constantly divided Stage III – Rooting & Hardening explant transferred to root media explant returned to soil

Methods of Micropropagation Organogenesis Organogenesis via callus formation Direct adventitious organ formation Embryogenesis Direct embryogenesis Indirect embryogenesis Microcutting Meristem culture (Mericloning) Bud culture

Organogenesis PGRs are prob. the most important factor affecting organogenesis cytokinins tend to stimulate formation of shoots auxins tend to stimulate formation of roots The central dogma of organogenesis: a high cytokinin:auxin ratio promotes shoots and inhibits roots a high auxin:cytokinin ratio promotes roots and/or callus formation while inhibiting shoot formation

Organogenesis The process of initiation and development of a structure that shows natural organ form and function. The ability of non-meristematic plant tissues to form various organs de novo. The production of roots, shoots or leaves. These organs may arise out of pre-existing meristems or out of differentiated cells. This, like embryogenesis, may involve a callus intermediate but often occurs without callus.

Somatic Embryos Tissue culture maintains the genetic of the cell or tissue used as an explant. Tissue culture conditions can be modified to cause to somatic cells to reprogram into a bipolar structure. These bipolar structures behave like a true embryo - called somatic embryos.

An Embryo is made up of actively growing cells and the term is normally used to describe the early formation of tissue in the first stages of growth.

Somatic Embryogenesis The process of initiation and development of embryos or embryo-like structures from somatic cells The production of embryos from somatic or “non-germ” cells. Usually involves a callus intermediate stage which can result in variation among seedlings

Somatic embryogenesis Somatic embryogenesis is a useful regeneration pathway for many monocots and dicots, but is especially useful for the grasses Types of embryogenesis zygotic embryogenesis – the result of normal pollination and fertilization somatic embryogenesis – embryos from (cultured) sporophytic cells , that is embryos arise indirectly

The composition of the culture medium controls the process- auxin (usually 2,4-D) added causes induction, the formation of embrygogenic clumps or proembryogenic masses (PEMs) (induction medium) auxin is deleted and the clumps become mature embryos (maturation medium)

Stages of development early cell division doesn't follow a fixed pattern, unlike with zygotic embryogenesis later stages are very similar to zygotic embryos (dicot pattern) globular stage (multicellular) heart-shaped stage (bilateral symmetry) – bipolarity torpedo-shaped stage – consists of initial cells for the shoot/root meristem

Stages of Somatic embryo development

Somatic Embryogenesis Stimulation of callus or suspension cells to undergo a developmental pathway that mimics the development of the zygotic embryo.

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.

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 Limitations Equipment/facility intensive operation Technical expertise in management positions Protocols not optimized for all species Liners may not fit industry standard Propagules may be too expensive

Applications Rapid increase of stock of new varieties. Elimination of diseases. Cloning of plant types not easily propagated by conventional methods. Propagules have enhanced growth features (multibranched character;Ficus, Syngonium)