Subculturing

Once a particular kind of organised or unorganised growth has been started in vitro, it will usually continue if callus cultures, suspension cultures, or cultures of indeterminate organs (see below) are divided to provide new explants for culture initiation on fresh medium. Subculturing often becomes imperative when the density of cells, tissue or organs becomes excessive; to increase the volume of a culture; or to increase the number of organs (e.g. shoots or somatic embryos) for micropropagation. The period from the initiation of a culture or a subculture to the time of its transfer is sometimes called a passage. The first passage is that in which the original explant or inoculum is introduced.

Suspensions regularly subcultured at the end of the period of exponential growth can often be propagated over many passages. However, many cultures reach a peak of cell aggregation at this time and aggregation often becomes progressively more pronounced in subsequent passages (Street, 1977b). Subculture is therefore more conveniently carried out during the stationary phase when cell aggregation is least pronounced. Rapid rates of plant propagation depend on the ability to subculture shoots from proliferating shoot or node cultures, from cultures giving direct shoot regeneration, or callus or suspensions capable of reliable shoot or embryo regeneration.

A further reason for transfer, or subculture, is that the growth of plant material in a closed vessel eventually leads to the accumulation of toxic metabolites and the exhaustion of the medium, or to its drying out. Thus, even to maintain the culture, all or part of it must be transferred onto fresh medium. Callus subcultures are usually initiated by moving a fragment of the initial callus (an inoculum) to fresh medium in another vessel. Shoot cultures are subcultured by segmenting individual shoots or shoot clusters. The interval between subcultures depends on the rate at which a culture has grown: at 25°C, subculturing is typically required every 4-6 weeks. In the early stages of callus growth it may be convenient to transfer the whole piece of tissue to fresh medium, but a more established culture will need to be divided and only small selected portions used as inocula. Regrowth depends on the transfer of healthy tissues.

Decontamination procedures are theoretically no longer necessary during subculturing, although sterile transfer procedures must still be used. However, when using shoot or node cultures for micropropagation, some laboratories do re-sterilise plant material at this stage as a precaution against the spread of contaminants (see Volume 2). Cultures which are obviously infected with micro-organisms should not be used for subculturing and should be autoclaved before disposal.

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