Micropropagation

[aayush_005]

Tissue culture is a term used for the growth of plants or more commonly plant parts in sterile culture. Micropropagation is a method of propagating plants using very small parts of plants that are grown in sterile culture. Micropropagation is not most likely the major use of tissue culture for molecular biologists or plant breeders. However, it is important commercially, and can be used to introduce several concepts that apply to all of tissue culture.

Georges Morel (1960) first showed the potential of clonal propagation in culture by describing in vitro multiplication of Cymbidium orchids. Small, spherical bodies, called protocorms, formed on cultured shoot tip explants. Several protocorms formed on each explant, and when divided and transferred, additional protocorms formed. The rate of multiplication was such that several million plants could be produced from one shoot tip in a single year. As a result, many of the economically important orchids are now usually propagated in vitro, although techniques have not been worked out for all orchid types.

However, credit for the development of the field of micropropagation in general goes without a doubt to Murashige, who showed that many plants, in addition to orchid, could be propagated in vitro. Micropropagation has been shown to be especially useful in horticultural crops.

Micropropagation is usually achieved by the release and growth of pre-existing (axillary or lateral) meristems in the initial culture; this is often referred to as shoot culture. A formal definition is clonal in vitro propagation by repeated enhanced formation of axillary shoots from shoot tips or lateral meristems following culture on a medium supplemented with plant growth regulators, in particular cytokinins. The shoots produced are rooted either in vitro or out of culture (ex vitro).


A. Shoot meristems

In plants, organ initiation and development continue throughout the life cycle. This is in contrast to animals in which all organs are laid down during embryogenesis. Furthermore, there is a plasticity in plant development that allows for development according to environmental conditions. Since all plant organs develop from meristems, these structures are where plant development and plant form ultimately is regulated. Meristems are formed during embryogenesis and consist of groups of undifferentiated cells that will initiate organ primordia during plant life. The apical shoot meristem give rises to the complete shoot of the plant and the apical root meristem is the origin of the root.

Different zones can be distinguished within the apical shoot meristem. Cells in the central zone are large and divide infrequently while the peripheral zone, the site of organogenesis, consists of small cells that divide fast and differentiate. The third zone is the rib zone giving rise to cells of the stem and forming the boundary against fully differentiated cells. Like cells in the peripheral zone the rib cells originate from the undifferentiated cells in the central zone. Apart from forming cells of the stem, the rib zone has been suggested to act as an organizing center for the shoot. In vegetative and inflorescence meristems the central cells play a role comparable to animal stem cells as they are characterized by their undifferentiated state and their ability to regenerate themselves as they give rise to daughter cells differentiating into specialized cell types.
In stems, mitosis in the apical meristem of the shoot apex (also called the terminal bud) produces cells that enable the stem to grow longer and, periodically, cells that will give rise to leaves. The point on the stem where leaves develop is called a node. The region between a pair of adjacent nodes is called the internode.
Shoot apical meristems are found at the terminals (tops) of shoots. Further down the shoot are axillary, or lateral, meristems. There is typically an axillary bud in each leaf axil on the shoot and each encloses a shoot tip. Each of these buds has the potential to become a shoot under the proper conditions. In vivo, the growth and development of axillary buds is frequently suppressed by the apical dominance of the apical meristem, which produces auxin. In vitro, cytokinins can be added to the medium to overcome apical dominance and release the growth of the axillary meristems.

B. Advantages of Shoot Culture

• Shoot production is reliable and consistent.
• Multiplication rates can be three-fold to eight-fold a month.
• Plants produced via shoot culture are usually true-to-type and uniform
• Allows propagation of periclinal chimeras

C. Disadvantages of Shoot Culture

• Cytokinins do not release apical dominance in all species.
• There may be a difference in results between juvenile and mature tissue of perennial species; shoot culture may require a reversion to juvenility.
• It may be difficult to root shoots – cytokinin “carry-over” effect.
• It may not be possible to get uniform shoot production in vitro, which can be very important in commercial operations.
• The procedure is relatively labor intensive, with high upfront costs to get started.


D. Applications of Micropropagation

• The obvious: clonal mass propagation of large numbers of uniform plants.
• Micropropagation may allow faster production of plants that are slow to propagate in vivo.
• It may decrease the time needed for bulk-up of new cultivars before they are introduced commercially.
• Storage of germplasm, e.g. by cryopresevation. More on this later.


E. Stages of Micropropagation

Micropropagation is now typically divided into 5 stages. Stages 1-4 were originally proposed by Murashige; Debergh and Maene added Stage 0.
Stage 0: Preparative Stage: Donor Plant Selection and Preparation

Explant quality and responsiveness is influenced the physiological phytosanitary condition of the donor plants.

• Donor (stock) plants indexed for pathogens.
• Pathogen-free stock plants maintained in clean conditions (low humidity, drip irrigation).
• Vigorous growth is encouraged, but not over-fertilization.
• Donor plants may be pretreated in certain ways.


Stage 1: Establishment of Explant in Culture

• Surface-sterilization – disinfestation: Must free explant tissues of all contaminating microorganisms, but not cause phytotoxity.
• Isolation of shoot tip under sterile conditions.
• Medium - Must contain all components necessary to nourish explant (medium composition) and to make the explant perform as desired (PGRs).
o Browning of the medium: Results from oxidation of phenolics leached out from cut surfaces of explants; often seen with adult woody species. Handled with anti-oxidants, frequent transfer.
o Medium formulation is often standard, e.g. M&S, but more complex media may be necessary for smaller explants.
o Medium may be semi-solid or liquid; there are advantages and disadvantages of each.
• Environmental conditions
o Light
o Temperature
o Relative humidity
• Culture stabilization.
Stage 2: Multiplication: Proliferation of Axillary Shoots

• Repeated enhanced axillary shoot production.
• Encouraged by cytokinin in the medium, alone or with a smaller amount of auxin. Amount of cytokinin and presence and amount of auxin must be determined empirically.
• Shoots harvested and shoot clusters transferred to fresh medium at frequent, regular intervals.
• Number of subcultures possible from the original culture varies with species/cultivar: reduction of growth, increase in mutations.


Stage 3: Pretransplant (Rooting)

• Adventitious rooting of shoots or shoot clusters in vitro.
• Harvested shoots may be pretreated before rooting: prehardening, elongation, fulfilling dormancy requirements.
• For root initiation in vitro, auxins are important.
• More dilute medium, activated charcoal may be added.
• Advantages of rooting after removal from culture
o Reduced costs
o Structurally and physiologically better
o Damage to roots may occur during transplanting


Stage 4: Transfer to Natural Environment:

Acclimatization: Process by which physiologically and anatomically adjust from in vitro to ex vitro conditions.
• Relatively slow process, may take weeks, starch reserves important.
• Must adjust from high to lower relative humidity (e.g. from 98-99% to 20 - 60%): development of sufficient defenses to control water loss.
o Poor cutilcle development: epicuticular wax needs to be formed.
o Abnormal stomatal development and function.
 Either are not properly depressed.
 Or do not open and close properly.
o Non- or poorly functional roots.
• From clean vs. presence of pathogens.
• Must adjust from low light to high light: from low photosynthetic competence (heterotrophic nutrition) to photosynthetic competence.
o Poorly differentiated leaf structure.
o Poorly developed chloroplasts.
o Supplied carbohydrate source to independent carbon fixation.
• There may be culture medium carryover effects.
• Dormancy may need to be overcome.
• Soil medium and container important.
• Acclimatization structures:
o Plastic covers.
o Humidity tent. Overhead mist.
o Fog system.