Micropropagation is the propagation of plants through tissue culture.

There are five stages to micro-propagation. These stages are:

  • Stage 0:   Donor Plant Selection
  • Stage I:   Establishment
  • Stage II:   Multiplication
  • Stage III:   Rooting
  • Stage IV:   Acclimatization

Stage I Establishment

Photo of stage 1 of micropropagation, establishment. Photo is of explant materials being estblished in micropropagation medium.

Stage II multiplication

Photo of stage II micropropagation, multiplication. Early stage seedlings are developing in microprogation medium.

Stage III Rooting

Photo of stage III of micropropagation, rooting. Well developed seedling in micropropagation medium, with roots developed.

Stage IV Acclimatization

Photo of stage IV micropropagation, acclimation. Seedlings have been transferred to covered tray with different growing medium.

Illustration showing and describing components of all five stages of micropropagation. Stage 0, Donor Plant Selection. Explant Source. Stage I, Establishment. Shoots are established in culture (2- to 3-months). Stage II, Shoot Multiplication. Subculture to muultiply microshoots (4- to 8-week cycles) Stage III, Root Formation. In vitro rooting (~ 4-weeks). Ex vitro rooting (~ 4- to 8-weeks). Stage IV, Acclimatization. Plants are gradually acclimatized to ambient environmental conditions and moved to unit cells. After Stage IV is nursery production.

Stage 0 - Donor Plant Selection

Source plants are manipulated prior to severance of explants.

Stage I - Establishment

During the establishment stage, the explant must be disinfested and stabilized.

The explant is usually sterilized with a combination of detergent and bleach. In difficult situations, alcohol or a fungicide may be used.

The objective of this stage is to get clean cultures that can begin the process of shoot multiplication.

Photo of explant material being disinfested in bleach.

Disinfesting explants in bleach

Photo of initial explants growing in micropropagation medium.

Initial explant

Micropropagation Stage II - Shoot multiplication

The objective of the shoot multiplication stage is to increase the number of shoots produced by the original explant.

By subculturing these new shoots on to new medium, the number of shoots produced in culture increases dramatically.

Photo of technician dividing explant shoots into subcultures.

The technician is preparing to subculture. The culture or a portion of the culture is removed from the jar and placed on a sterile paper towel.

Photo of technician placing culture onto a sterile paper towel.

Photo of technician dividing culture into multiple subcultures.

A scalpel and forceps are used to cut and separate the larger culture into smaller pieces for transfer to a new jar to complete the subculturing procedure.

Photo of technician continuing to divide a culture using scalpel and forceps.

Photo of technician beginning to place divided subcultures into a new jar.

Click on the button below to see Stage II movie.

A high cytokinin to auxin ratio is used during the multiplication stage to induce axillary or adventitious shoot formation.

This ratio is decided upon by preliminary research.

Too high a concentration of cytokinin will result in a high number of adventitious shoots that do not elongate.

Common cytokinins used in culture are benzyladenine and kinetin.

A chart showing the relationship between Cytokinin concentration and Auxin concentration. Three points are shown; a low auxin/high cytokinin combination for adventitious shoot formation, a moderate auxin/moderate cytokinin combination for a callus, and a high auxin/low cytokinin combination for a plantlet showing root formation.

Micropropagation Stage III - Root formation

Shoots multiplied in culture must be rooted in Stage III in order to create a new plantlet.

In the rooting stage, microcuttings are induced to form roots - usually by application of auxin.

In general, species root easier in tissue culture than they do from conventional cuttings.

Photo of a plantlet with roots forming.

Microcuttings can either be rooted in vitro or ex vitro.

In general, microcuttings rooted ex vitro have a more normal root system and acclimatize to a normal growing environment better than cuttings rooted in vitro.

However, the propagator has more control over the rooting environment in vitro and this method may fit their production scheme better.

Photo of a plantlet being grown in vitro in gel micropropagation medium.

in vitro

Photo of a plantlet being grown ex vitro in a cell of rooting substrate.

ex vitro

Microcuttings are inserted directly into the rooting substrate often using forceps to handle the small cuttings.

Photo of technician using forceps to insert microcuttings into trays with cells of rooting substrate.

Photo showing detail of tray with waiting cells and those with microcuttings already inserted.

At each work station the technicians have their rooting flats, a syringe bottle to spray microcuttings periodically to keep them from drying out.

Photo of technicians sticking microcuttings at a workstation.

Technicians sticking microcuttings at a workstation.

Photo of microcuttings after the agar has been washed off.

Microcuttings after the agar has been washed off.

Photo of sticking microcuttings with forceps.

Sticking microcuttings.

Click on the button below to see Stage III movie.

Micropropagation Stage IV - Acclimatization

Finally, after roots have become well established on the microcutting, plantlets must be acclimatized to a normal growing environment in Stage IV.

Photo of plantlets growing in a covered rooting flat.

This involves gradually moving to open-air conditions where the humidity is reduced and the light levels increased.

This is a vulnerable stage for plantlet survival where the propagator can see large losses without proper acclimatization.

Photo of rooted microcuttings being acclimatized using intermittent mist in a closed polyethylene tent.

Rooted microcuttings being acclimatized using intermittent mist in a closed polyethylene tent.