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      The most critical aspect of in vitro culture is the provision of a sterile environment. A plant has certain natural defenses against pathogens and the abiotic environment in which it grows. Cells and tissues lack such protection once extracted from the parent plant. The environment for growing plants in the soil under natural conditions should provide adequate moisture, nutrients, light, temperature, and air. Plant performance can be enhanced by supplementing the growth environment (e.g. by fertilization, irrigation). In tissue and cell culture, plant materials are grown in a totally artificial environment in which nutrients, plus additional factors (e.g. growth regulators) and sometimes antibacterial substances, are supplied. The cultural environment in tissue culture may be adjusted by the researcher to control the growth and development of the cultured material. For example, the researcher may modify the hormonal balance in the culture medium to favor only root or only shoot development. The components of a tissue culture medium may be categorized into four groups: mineral elements, organic compounds, growth regulators, and a physical support system.

7.12 Micropropagation

      Seed is the preferred propagule for use in the propagation and cultivation of most agronomic species. This is because they are easy to handle before and during the production of the plant, and seeds of most species can be stored for many years or decades. However, a number of major food crops and horticultural species are vegetatively (asexually) propagated as a preferred method, because of biological reasons (e.g. self‐incompatibility) and the lack of uniformity in seed progeny. Micropropagation is the in vitro clonal propagation or reproduction of plants. It is used more commonly for commercial propagation of ornamentals and other high‐priced horticultural species than for field crop species. Micropropagation can utilize preexisting meristems (specific regions where cells are undifferentiated or have no specific assigned roles or function) or nonmeristematic tissue. The common methods of micropropagation may be divided into three categories: (i) axillary shoot production, (ii) adventitious shoot production, and (iii) somatic embryogenesis.

      Micropropagation may be summarized in five general steps:

      1 Selection of explantThe plant part (e.g. meristem, leaf, stem tissue, buds) to initiate tissue culture is called the explant. It must be in good physiological condition and disease‐free. Factors that affect the success of the explant include its location on the plant, age, or developmental phase. Explants that contain shoot primordia (e.g. meristems, node buds, shoot apices) are preferred. Also, explants from younger (juvenile) plants are more successfully used in micropropagation.

      2 Initiation and aseptic culture establishmentThe explant is surface sterilized (e.g. with bleach, alcohol) before placing on the medium. Small amounts of plant growth regulators may be added to the medium for quick establishment of the explant.

      3 Proliferation of axillary shootsAxillary shoot proliferation is induced by adding cytokinin to the shoot culture medium. Cytokinin to auxin ratio of about 50 : 1 produces shoot with minimum callus formation. New shoots may be subcultured at an interval of about four weeks.

      4 RootingAddition of auxin to the medium induces root formation. Roots must be induced on the shoot to produce plantlets for transfer into the soil. It is possible to root the shoot directly in the soil.

      5 Transfer to natural environmentBefore transferring into the field, seedlings are gradually moved from ideal lab conditions to more natural climate room or greenhouse conditions by reducing the relative humidity and increasing light intensity, a process called hardening off.

      7.12.1 Axillary shoot production

      Preexisting meristems are used to initiate shoot culture (or shoot‐tip culture). The size of the shoot tip ranges between 1 and 10 mm long. Cytokinin is used to promote axillary shoot proliferation. Some species (e.g. sweet potato) do not respond well to this treatment. Instead, shoots consisting of single or multiple nodes per segment are used. These explants are placed horizontally on the medium and from them single unbranched shoots arise that may be induced to root to produce plantlets.

      Shoot tips are easy to excise from the plant and are genetically stable. They contain pre‐formed incipient shoot and are phenotypically homogeneous. These explants have high survival and growth rates. Axillary and terminal buds have the advantages of shoot tips, but they are more difficult to disinfect. On the other hand, meristem tips contain preformed meristems and are genetically stable and phenotypically