Problems Of Establishment

2.5.1. Phenolic oxidation

Some plants, particularly tropical species, contain high concentrations of phenolic substances that are oxidised when cells are wounded or senescent. Isolated tissue then becomes brown or black and fails to grow. The prevention of blackening, which can be a serious problem in tissue culture, is discussed in Chapter 11.

2.5.2. Minimum inoculation density

Certain essential substances can pass out of plant cells by diffusion. Substances known to be released into the medium by this means include alkaloids, amino acids, enzymes, growth substances and vitamins (Street, 1969). The loss is of no consequence when there is a large cluster of cells growing in close proximity or where the ratio of plant material to medium is high. However, when cells are inoculated onto an ordinary growth medium at a low population density, the concentration of essential substances in the cells and in the medium can become inadequate for the survival of the culture. For successful culture initiation, there is thus a minimum size of explant or quantity of separated cells or protoplasts per unit culture volume. Inoculation density also affects the initial rate of growth in vitro. Large explants generally survive more frequently and grow more rapidly at the outset than very small ones. In practice, minimum inoculation density varies according to the genotype of plant being cultured and the cultural conditions. For commencing suspension cultures it is commonly about 1-1.5 x 104 cells/ml.

The minimum cell density phenomenon is sometimes called a 'feeder effect' because deficiencies can often be made up by the presence of other cells growing nearby. Suspension cultures can be started from a low density of inoculum by 'conditioning' a freshly prepared medium - i.e. allowing products to diffuse into it from a medium in which another culture is growing actively, or adding a quantity of filter-sterilised medium which has previously supported another culture. The use of conditioned media can reduce the critical initial cell density by a factor of about 10 (Stuart and Street, 1969).

It is possible to overcome the deficiencies of plant cells at low starting densities by adding small amounts of known chemicals to a medium. For example, Kao and Michayluk (1975) have shown that Vicia hajastana cells or protoplasts can be cultured from very small initial inocula or even from individual cells: a standard culture medium was supplemented with growth regulators, several organic acids, additional sugars (apart from sucrose and glucose), and in particular, casein hydrolysate (casamino acids) and coconut milk.

There is often a maximum as well as a minimum plating or inoculation density for plant cells or protoplasts. In a few cases the effective range has been found to be quite narrow. Some effects of inoculation density on morphogenesis are described in Chapter 10.

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