Plant Tissue Culture Procedure - Background

1. INTRODUCTION
Plant tissue culture is the science of growing plant cells, tissues or organs isolated from the mother plant, on artificial media. It includes techniques and methods used to research into many botanical disciplines and has several practical objectives. Before beginning to propagate plants by tissue culture methods, it is necessary to have a clear understanding of the ways in which plant material can be grown and manipulated in ‘test tubes’. This chapter therefore describes the techniques that have been developed for the isolation and in vitro culture of plant material, and shows where further information can be obtained. Both organised and unorganised growth are possible in vitro.

1.1. ORGANISED GROWTH
Organised growth contributes towards the creation or maintenance of a defined structure. Itoccurs when plant organs such as the growing points of shoots or roots (apical meristems), leaf initials, young flower buds or small fruits, are transferred to culture and continue to grow with their structure preserved. Growth that is coherently organised also occurs when organs are induced. This may occur in vitro either directly upon an organ or upon a piece of tissue placed in culture (an explant), or during the culture of previously unorganised tissues. The process of de novo organ formation is called organogenesis or morphogenesis (the development of
form).

1.2. UNORGANISED GROWTH
The growth of higher plants depends on the organised allocation of functions to organs which in consequence become differentiated, that is to say, modified and specialised to enable them undertake their essential roles. Unorganised growth is seldom found in nature, but occurs fairly frequently when pieces of whole plants are cultured in vitro. The cell aggregates, which are then formed, typically lack any recognisable structure and contain only a limited number of the many kinds of specialised and differentiated cells found in an intact plant. A differentiated cell is one that has developed a specialised form (morphology) and/or function (physiology). A differentiated tissue (e.g. xylem or epidermis) is an aggregation of differentiated cells. So far, the formation of differentiated cell types can only be controlled to a limited extent in culture. It is not possible, for example, to maintain and multiply a culture composed entirely of epidermal cells. By contrast, unorganised tissues can be increased in volume by subculture and can be maintained on semisolid or liquid media for long periods. They can often also be used to commence cell suspension cultures. Differentiation is also used botanically to
describe the formation of distinct organs through morphogenesis.

2. TISSUE CULTURE

2.1. CULTURES OF ORGANISED STRUCTURES
Organ culture is used as a general term for those types of culture in which an organised form of growth can be continuously maintained. It includes the aseptic isolation from whole plants of such definite structures as leaf primordia, immature flowers and fruits, and their growth in vitro. For the purposes of plant propagation, the most important kinds of organ culture are:
• Meristem cultures, in which are grown very small excised shoot apices, each consisting of the apical
meristematic dome with or without one or two leaf primordia. The shoot apex is typically grown to give one single shoot.
• Shoot tip, or shoot cultures, started from excised shoot tips, or buds, larger than the shoot apices employed to establish meristem cultures, having several leaf primordia. These shoot apices are usually cultured in such a way that each produces multiple shoots.
• Node cultures of separate lateral buds, each carried on a small piece of stem tissue; stem pieces carrying either single or multiple nodes may be cultured. Each bud is grown to provide a single shoot.
• Isolated root cultures. The growth of roots, unconnected to shoots: a branched root system may be obtained.

• Embryo cultures, where fertilised or unfertilised zygotic (seed) embryos are dissected out of developing seeds or fruits and cultured in vitro until they have grown into seedlings. Embryo culture is quite distinct from somatic embryogenesis (see below). These types of cultures are described in more detail later in this chapter.

2.2. CULTURES OF UNORGANISED TISSUES
‘Tissue culture’ is commonly used as a collective term to describe all kinds of in vitro plant cultures although strictly it should refer only to cultures of unorganised aggregates of cells. In practice the following kinds of cultures are most generally recognised:
• Callus (or tissue) cultures. The growth and maintenance of largely unorganised cell masses,  which arise from the uncoordinated and disorganised growth of small plant organs, pieces of plant tissue or previously cultured cells.
• Suspension (or cell) cultures. Populations of plant cells and small cell clumps, dispersed in an agitated, that is aerated, liquid medium.
• Protoplast cultures. The culture of plant cells that have been isolated without a cell wall.
• Anther cultures. The culture of complete anthers containing immature pollen microspores. The objective is usually to obtain haploid plants by the formation of somatic embryos (see below) directly from the pollen, or sometimes by organogenesis via callus. Pollen cultures are those initiated from pollen that has been removed from anthers.

2.3. USING TISSUE CULTURES FOR PLANT PROPAGATION
The objective of plant propagation via tissue culture, termed micropropagation, is to propagate plants true-to-type, that is, as clones. Plants obtained from tissue culture are called microplants and can be derived from tissue cultures in three ways:
• from pre-existing shoot buds or primordial buds (meristems) which are encouraged to grow and proliferate;
• following shoot morphogenesis when new shoots are induced to form in unorganised tissues or directly
upon explanted tissues of the mother plant;
• through the formation of somatic embryos which resemble the seed embryos of intact plants, and which
can grow into seedlings in the same way. This process is called somatic embryogenesis. To obtain plants by the first two of these methods, it is necessary to treat shoots of an adequate size as miniature cuttings and induce them to produce roots. The derivation of new plants from cells, which would not normally have taken part in the process of regeneration, shows that living, differentiated plant cells may express totipotency, i.e. they each retain a latent capacity to produce a whole plant. Totipotency is a special characteristic of cells in young tissues and meristems. It can be exhibited by some differentiated cells, e.g. cambial cells and leaf palisade cells but not those which have developed into terminally differentiated structures (e.g. sieve tubes or
tracheids). Theoretically, plant cells, organs, or plants, can all be cloned, i.e., produced in large numbers as a
population where all the individuals have the same genetic constitution as the parent. Present tissue culture techniques do not permit this in every case and irregularities do sometimes occur, resulting in ‘somaclonal variants’ (Larkin and Scowcroft, 1981). Nevertheless, as will be described in the chapters, which follow, a very large measure of success can be achieved and cultures of various kinds can be used to
propagate plants.