Initiating Tissue Cultures

2.4.1. Explants

Tissue cultures are started from pieces of whole plants. The small organs or pieces of tissue that are used are called explants. The part of the plant (the stock plant or mother plant) from which explants are obtained, depends on:

• the kind of culture to be initiated;

• the purpose of the proposed culture;

Explants can therefore be of many different kinds. The correct choice of explant material can have an important effect on the success of tissue culture. Plants growing in the external environment are invariably contaminated with micro-organisms and pests. These contaminants are mainly confined to the outer surfaces of the plant, although, some microbes and viruses may be systemic within the tissues (Cassells, 1997). Because they are started from small explants and must be grown on nutritive media that are also favourable for the growth of microorganisms, plant tissue cultures must usually be established and maintained in aseptic conditions. Most kinds of microbial organism, and in particular bacteria and fungi, compete adversely with plant material growing in vitro. Therefore, as far as possible, explants must be free from microbial contaminants when they are first placed on a nutrient medium. This usually involves growing stock plants in ways that will minimise infection, treating the plant material with disinfecting chemicals to kill superficial microbes, and sterilising the tools used for dissection and the vessels and media in which cultures are grown (for a review see Cassells and Doyle, 2005). Some kinds of plants can, however, be micropropagated in non-sterile environments (see Chapter 3).

2.4.2. Isolation and incubation

The work of isolating and transferring cultured plant material is usually performed in special rooms or inside hoods or cabinets from which microorganisms can be excluded. Cabinets used for isolation can be placed in a draught-free part of a general laboratory, but are much better situated in a special inoculation or transfer room reserved for the purpose. The accommodation, equipment and methods that are required for successful inoculation and transfer are described in Volume 2. Cultures, once initiated, are placed in incubators or growth rooms where lighting, temperature and humidity can be controlled. The rate of growth of a culture will depend on the temperature (and sometimes the lighting) regime adopted.

2.4.3. The cultural environment

Plant cultures are commenced by placing one or more explants into a pre-sterilised container of sterile nutrient medium. Some explants may fail to grow, or may die, due to microbial contamination: to ensure the survival of an adequate number, it therefore is usual to initiate several cultures at the same time, each being started from an identical organ or piece of tissue. Explants taken from stock plants at different times of the year may not give reproducible results in tissue culture. This may be due to variation in the level of external contaminants or because of seasonal changes in endogenous (internal) growth regulator levels in the stock plant (see Chapter 11).

Plant material will only grow in vitro when provided with specialised media. A medium usually consists of a solution of salts supplying the major and minor elements necessary for the growth of whole plants, together with:

• various vitamins (optional);

• various amino acids (optional);

• an energy source (usually sucrose).

The components of plant tissue culture media are discussed in Chapters 3 and 4. The compositions of specific media are described in Volume 2. Growth and development of plant cultures usually also depends on the addition of plant growth regulators to the medium (see Chapters 5, 6 and 7). Plant growth regulators are compounds, which, at very low concentration, are capable of modifying growth or plant morphogenesis. Many workers define a medium as a completed mixture of nutrients and growth regulators. This is a rather inflexible method, as growth regulators frequently need to be altered according to the variety of plant, or at different stages of culture, whilst the basic medium can stay unchanged. It is therefore recommended that nutritional and regulatory components should be listed separately. Plant material can be cultured either in a liquid medium or on a medium that has been partially solidified with a gelling agent (see Chapter 4). The method employed will depend on the type of culture and its objective.

2.4.5. Solidified media

Media which have had a gelling agent added to them, so that they have become semi-solid, are widely used for explant establishment; they are also employed for much routine culture of callus or plant organs (including micropropagation), and for the long-term maintenance of cultures. Agar is the most common solidifying agent, but a gellan gum is also widely used (Chapter 4).

Cultures grown on solid media are kept static. They require only simple containers of glass or plastic, which occupy little space. Only the lower surface of the explant, organ or tissue is in contact with the medium. This means that as growth proceeds there may be gradients in nutrients, growth factors and the waste products of metabolism, between the medium and the tissues. Gaseous diffusion into and out of the cells at the base of the organ or tissue may also be restricted by the surrounding medium.

2.4.6. Liquid media

Liquid media are essential for suspension cultures, and are preferred for critical experiments on the nutrition, growth and cell differentiation in callus tissues. They are also used in some micropropagation work. Very small organs (e.g. anthers) are often floated on the top of liquid medium and plant cells or protoplasts can be cultured in very shallow layers of static liquid, providing there is sufficient gaseous diffusion. Larger organs such as shoots (e.g. proliferating shoots of shoot cultures) can also often be grown satisfactorily in a shallow layer of non-agitated liquid where part of the organ protrudes above the surface. However, some method of support is necessary for small organs or small pieces of tissue, which would otherwise sink below the surface of a static liquid medium, or they will die for lack of aeration. Systems of support which have been found to be effective and which can be used instead of agar-solidified media are described in Chapters 4.

Many tissues and organs, small and large, also grow well unsupported in a liquid medium, providing it is aerated by shaking or moving (see below). Some kind of agitation is essential for suspension cultures to prevent cells and cell aggregates settling to the bottom of the flask. Other purposes served by agitation include: the provision of increased aeration, the reduction of plant polarity, the uniform distribution of nutrients and the dilution of toxic explant exudates (Lim-Ho, 1982).

There are several alternative techniques. Plant cell suspensions can be cultured very satisfactorily when totally immersed in a liquid culture medium, providing it is shaken (by a rotary or reciprocating shaking machine) or stirred (e.g. by a magnetic stirrer) to ensure adequate aeration. This method may also be used for culturing organs of some plants (e.g.

proliferating shoot cultures), but the fragmentation, which occurs, can be disadvantageous.

Periodic immersion may be achieved by growing cultured material in tubes or flasks of liquid medium which are rotated slowly. Steward and Shantz (1956) devised so-called 'nipple flasks' for this purpose which had several side-arms. They were fixed to a wooden wheel, which was rotated so that tissue in the arms of each flask was alternately bathed in medium and drained or exposed to the air (Fig. 1.1). This technique ensured that callus tissue for which they were used was well aerated. The medium usually became turbid as cells dissociated from the callus and started a cell suspension. Flasks of this sort are seldom used to-day because of their cost. A similar alternating exposure can be achieved by placing calluses in vessels, which are rotated slowly.

An alternative to the costly rotating systems to achieve periodic immersion of the cultures, is the increasingly popular temporary immersion system in which static vessels are periodically or temporarily flooded with culture medium (Fig. 1.2; Teisson and Alvard, 1995). Medium is pumped from a reservoir container into the culture vessel for experimentally determined time intervals repeated over a 24 hour cycle. This system prevents anoxia and has the advantage that the medium can easily be changed in the reservoir.

Fig. 1.1 A nipple flask for growing callus in a liquid medium.
Fig. 1.3 An illustration of micropropagation in a bioreactor. a. air inlet; b. sparger; c. raft supporting explants; d. air outlet; e. membrane filter. The bioreactor culture is initiated by inoculation with nodes or buds from conventional agar culture. For details of bioreactor design see Fig. 1.9.

Liquid medium in flasks or column bio-reactors forces within air-lift reactors are much less than in (fermentors) can be circulated and at the same time mechanically-stirred vessels so that plant cell aerated, by the introduction of sterile air. Shearing suspensions suffer less damage. Bio-reactors are used in the pharmaceutical industry to produce high value plant secondary products and to carry out substrate conversions. Low cost bio-reactors developed for micropropagation have been described in detail in Hvoslef-Eide and Preil (2005) (Fig. 1.3).

Rather than immersing callus or organ cultures, liquid medium may be slowly dripped onto the growing tissues or applied as a mist and afterwards the liquid drained or pumped away for recirculation (Weathers and Giles, 1987). A particular advantage of this technique is the ability to grow cultures in a constant and non-depleted medium; nutrients can be varied frequently and rapidly and their availability controlled by altering either concentration or flow rate. Toxic metabolites, which in a closed container might accumulate and inhibit growth, can be removed continuously. As complicated apparatus is needed, the method has not been widely used.

The relative merits of solid and liquid media (and combinations of both) are discussed further in Chapter 12.

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