Stages Of Micropropagation

Professor Murashige of the University of California (Riverside) defined three steps or stages (I-III) in the in vitro multiplication of plants (Murashige, 1974). These have been widely adopted by both research and commercial tissue culture laboratories because they not only describe procedural steps in the micropropagation process, but also usually represent points at which the cultural environment needs to be changed.

Some workers have suggested that the treatment and preparation of stock plants should be regarded as a separately numbered stage or stages. We have adopted the proposal of Debergh and Maene (1981) that such preparative procedures should be called Stage 0. A fourth stage (IV), at which plants are transferred to the external environment, is now also commonly recognised. A general description of Stages 0-IV is therefore provided below, while the manner in which Stages I-III might be applied to different methods of micropropagation is given in Table 2.1.

Table 2.1 Stages in the available methods of micropropagation.

Methods of Micropropagation

Stage of culture

I. Initiating a culture

Growth of excited tissues/organs in vitro free from algae, bacteria, fungi and other contaminants.

II. Increasing propagules

Inducing the cultures to produce numbers of shoots or somatic embryos.

III. Preparation for soil transfer

Separating and preparing propagules to have a high rate of survival as individual plants in the external environment.

Shoot Cultures

Shoots from floral meristems

Multiple shoots from seeds

Transfer of disinfected shoot tips or lateral buds to solid or liquid media and the commencement of shoot growth to ca. 10mm.

Aseptic isolation of pieces of compound floral meristems.

Aseptic germination of seeds on a high cytokinin medium.

Induce multiple (axillary) shoot formation and growth of the shoots to a sufficient size for separation, either as new Stage II explants or for passage to III.

Inducing the many meristems to produce vegetative shoots, then as shoot tip culture.

Inducing multiple shoot proliferation. Shoot subculture

Elongation of buds formed at Stage II to uniform shoots. Rooting the shoots in vitro or outside the culture vessel.

As for shoot tip cultures. As for shoot tip cultures.

Meristem culture

Transfer of very small shoot tips (length 0.2-0.5mm) to culture. Longer shoot tips (1-2mm) can be used as explants if obtained from heat treated plants.

Growth of shoots to ca. 10mm, then as shoot tip culture, or as shoot multiplication omitted and shoots transferred to Stage III.

As for shoot tip cultures.

Node culture

As for shoot tip culture but shoots grown longer to show clear internodes.

Propagation by inducing the axillary bud at each node to grow into a single shoot. Subculturing can be repeated indefinitely.

As for shoot tip cultures.

Direct shoot regeneration from explants

Establishing suitable explants of mother plant tissue (e.g. leaf or stem segments) in culture without contamination.

The induction of shoots directly on the explant with no prior formation of callus. Shoots so formed can usually be divided and used as explants for new Stage II subcultures or shoot tip culture.

As for shoot tip cultures.

Direct embryogenesis

Establishing suitable embryogenic tissue explants or previously-formed somatic embryos.

The direct induction of somatic embryos on the explants without prior formation of callus.

Growth of the embryos into plantlets which can be transferred to the outside environment.

Indirect shoot regeneration from morphogenic callus

Initiation and isolation of callus with superficial shoot meristems.

Repeated subculture of small callus pieces followed by transfer to a shoot-inducing medium. The growth of shoots ca. 10mm in length.

Individual shoots are grown and rooted.

Indirect embryogenesis from embryogenetic callus or suspension cultures

Initiation and isolation of callus with the capacity to form somatic embryos, OR obtaining embryogenic suspension cultures from embryogenic callus or by de novo induction.

Subculture of the embryogenic callus or suspension culture followed by transfer to a medium favouring embryo development.

Growth of the somatic embryos into "Seedlings".

Storage organ formation

Isolation and culture of tissue/organs capable of forming storage organs.

Inducing the formation of storage organs and sometimes dividing them to start new Stage II cultures.

Growing shoots/plantlets obtained from storage organs for transfer to soil: OR growing the storage organs themselves to a size suitable for soil planting.

Requirements for the completion of each stage of micropropagation vary according to the method being utilised; the progress of cultures will not always fit readily into neat compartments. Furthermore, it is not always necessary to follow each of the prescribed steps. The stages are therefore described here for general guidance but should not be applied too rigidly.

Practices adopted at the various stages of micropropagation are discussed throughout the book, but are particularly mentioned in Volume 2.

Fig. 2.1 The principal methods of micropropagation.

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