Plant secondary metabolism is very important in determining flower colour, flavour of food, and plant resistance against pests and diseases. Moreover, it is the source of many useful chemicals such as drugs, dyes, flavours and fragrances (Verpoorte et al., 1999, 2000), which are the main active constituents or at least simple components of most medicinal plants and herbal dietary supplements. Therefore, it is of interest to be able to engineer the secondary metabolite production of the plant cell factory, e.g. to produce more of a useful chemical, to produce less of a toxic compound, or even to make new compounds or valuable herbal products. Our limited knowledge of secondary metabolite pathways and the genes involved is one of the main bottlenecks (Verpoorte et al., 2000).
Plant tissue culture techniques and genetic engineering of plants may be regarded as the main tools of plant biotechnology aimed at the production of plants with improved yield and agronomic traits, which means also improved products. The reader should keep in mind that each of these techniques has been derived from different biological disciplines, whose combination is essential for research advancement (Ver-poorte et al., 1999).
Plant cell tissue culture (PCTC) is the cultivation of plant cells or tissues on specially formulated nutrient media. In appropriate conditions, an entire plant can be regenerated from each single cell, permitting the rapid production of many identical plants. This technique finds different applications, including:
• micropropagation of high-value plants for nurseries when a high standard of plant quality is requested, as in the case of varieties with elite agro-metabolic traits;
• production of cell suspension cultures to be maintained in fermenter vessels with the aim of producing valuable products such as phytochemicals, enzymes and natural food flavourings;
• conservation via shoot-tip culture as well as through cryoconservation of cells and tissues of those plant varieties that cannot be maintained in a normal seed bank;
• production of valuable hybrid plants through embryo rescue and plant recovery from crosses between genetically distant parents;
• regeneration of whole plants after genetic manipulation of cell or tissue cultures.
Genetic engineering is the controlled modification of genetic material (DNA) by artificial means. It relies upon the isolation of specific stretches of DNA using specialized enzymes, which cut the DNA at precise locations. Selected DNA fragments can then be transferred into plant cells in several ways:
• Agrobacterium technology, the best-established gene-transfer method for plants, employs this pathogenic soil bacterium as a vector so that desirable genetic information rather than that which induces the plant disease is transferred into plants (Zambryski, 1992).
• Bio-ballistic methods involve sticking the DNA that is to be introduced into the plant on to minute gold or tungsten particles, then firing these (like bullets) into the plant tissue. A proportion of the plant cells treated in this way take up the DNA from the metal pellets. Whole plants are then regrown from the cells by tissue culture (Newell, 2000).
• Electroporation is a technique which works best with plant tissues that have no cell walls (protoplasts or pollen tubes). Micro- to millisecond pulses of a strong electric field cause minute pores to appear momentarily in the plant cells, allowing DNA to enter from a surrounding solution. Recently this technique has also been applied to intact plant tissues (Chowrira et al., 1995). A more recent yet similar method uses microscopic silicious fibres to puncture holes in the plant cells (Songstad et al, 1995).
• Microinjection is based on the use of glass micropipettes (internal diameter 0.5—10 fxm) to effect the direct transfer of macromolecules into the cytoplasm or the nucleus of a recipient cell or cell structure (Draper and Scott, 1991).
• Somatic hybridization via protoplast fusion is a technique which is used to speed up the process and improve the precision of plant breeding, allowing in vitro cell fusion, producing hybrid plants between phylogenetically unrelated parent plants (Grosser et al, 2000).
• Somaclonal variation (Larkin and Scowcroft, 1983) is the genetic variation induced in plants regenerated via tissue culture methods. It can result in a range of genetically stable mutated clones, useful in crop improvement (Jain, 2001).
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