George Acquaah - Principles of Plant Genetics and Breeding

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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.

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. Micropropagationis 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.

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 homogeneous, but are more difficult to extract from the plant. Further, they have low survival rates. Meristems in general tend to be free of virus infection, even if the rest of the plant is infected. Meristems may therefore be the ideal explant to cure virus infected valuable clones.

Adventitious shoots originate from adventitious meristems. Non‐meristematic tissue can be induced to form plant organs (e.g. embryos, flowers, leaves, shoots, roots). Differentiated plant cells (with specific functional roles) can be induced to dedifferentiate from their current structural and functional state, and then embark upon a new developmental path to produce new structures. Adventitious shoot production through organogenesis occurs by one of two pathways – indirector direct.

1 Indirect organogenesisThe indirect organogenetic pathway goes through a stage in which a mass of dedifferentiated cells (callus) forms (i.e. the explant forms a callus from which adventitious meristems are induced and from which plant regeneration is initiated). The callus consists of an aggregation of meristem‐like cells that are developmentally plastic (can be manipulated to redirect morphogenic end point). The negative side of this method is that the callus phase sometimes introduces mutations (somaclonal variation, making this not always a 100% clonal procedure). The callus phase also makes it more technically challenging than shoot tip micropropagation.

2 Direct organogenesisDirect organogenesis bypasses a callus stage in forming plant organs. The cells in the explant act as direct precursors of a new primordium. This pathway is less common than the callus mediated pathway.

A zygote is formed after an egg has been fertilized by a sperm. The zygote then develops into an embryo ( zygotic embryo). In vitro tissue culture techniques may be used to induce the formation of embryos from somatic tissue ( non‐zygotic embryoor somatic embryogenesis) using growth regulators. Somatic embryos arise from a single cell rather than budding from a cell mass as in zygotic embryos. This option is very important in biotechnology since transgenesis in plants may involve the manipulation of single somatic cells. However, without successful regeneration, plant transformation cannot be undertaken. Somatic embryogenesis has been extensively studied in Apiaceae, Fabaceae, and Solanaceae.