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In this article, we will discuss about the conditions and stages of tissue culture.
Conditions of Tissue Culture:
I. Aseptic Conditions in Laboratory:
The tissue culture laboratory should have aseptic conditions. It means it should be well sterilized against pathogens.
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A pathogen free environment will help in maintaining good health of the callus, cell or protoplast cultures resulting in recovery of healthy plants from such cultures. The explant and glassware should be properly sterilized before their entry into the tissue culture laboratory.
II. Control of Temperature:
Air conditioning of the tissue culture laboratory is essential. Generally, temperature between 18 – 25°C is required. However, this varies from species to species. High temperature adversely affects the growth of callus.
III. Proper Culture Medium:
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Culture media have been developed by various workers for different crop species. The medium has to be modified as per the requirement of a species. These days, the culture media developed by Murashige and Skoog (1962) and Gamberg et. al. (1968) are used with some modifications in various crop species. This medium contains micro-and macro-nutrients, vitamins and hormones.
Stages of Tissue Culture:
Tissue culture can be broadly divided into four stages:
(i) During the first stage, suitable plant parts (called explants) are cut into small pieces, surface sterilized with specific anti-microbial chemicals and then inoculated on semi-solid culture media. The media generally contains inorganic macro and micro-nutrients vitamins, organic nitrogen, sugars, growth regulators etc. besides 5-10 per cent agar as gelling agent. These cultures are then kept usually at room temperature under continuous light or dark conditions, depending on the nature of the growing culture.
(ii) In the second stage, a mass of undifferentiated cells (called callus) is formed after incubation for 2-4 weeks which is further sub-cultured by transferring the callus to fresh medium called multiplication medium and incubated at room temperature (Fig. 1.7). This procedure can be repeated after every four weeks. During subculture, only a part of the culture from a vessel is transferred into the new culture vessel. Sub culturing is essential to maintain good health of the callus or tissues, because after some time some nutrients are depleted in the culture media and change of media becomes essential.
(iii) The third stage includes callus transfer to suspension cultures which contain all the necessary nutrients except the gelling agent. This is followed by differentiation which is induced by either allowing the callus pieces or suspension cultures to grow in specific media and within three to four weeks, numerous shoot buds or embryos appear which germinate into plantlets.
Details of Callus Culture and Suspension Culture:
In callus culture, cell division in explant forms a callus. Callus is irregular, unorganized and undifferentiated mass of actively dividing cells. Darkness and solid medium gelled by agar stimulates callus formation. The medium ordinarily contains the auxin, 2, 4-D, and often a cytokinin like BAR Both are growth regulators. This stimulates cell division in explant. Callus is obtained within 2-3 weeks.
A suspension culture consist of single cells and small groups of cells suspended in a liquid medium. Usually, the medium contains the auxin 2, 4-D. Suspension cultures must be constantly agitated at 100-250 rpm (revolutions per minute). Suspension cultures grow much faster than calls culture (Fig. 1.8).
The callus and suspension cultures may be used to achieve cell biomass production, regeneration of plantlets, production of transgenic plants and isolation of protoplasts.
(iv) In the last stage (Regeneration of Plantlets). Once the plantlets develop to a height of 5-6 inches, they are hardened and then transferred to soil. During hardening, plantlets are kept under reduced light and high humidity.
Hardening procedures make the plantlets capable of tolerating the relatively harsher environments outside the culture vessels. Besides this general scheme, different plant parts used as explants involve different procedures for the complete regeneration of a plantlet.
The various tissues used as explants could involve seeds, root and shoot tips, cut leaves, stem cuttings, protoplasts, anther, pollen grains, ovules, and axillary shoot buds.
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Plantlets can be obtained from cultured cells by two different ways:
(i) Shoot regeneration followed by rooting of the shoots, and
(ii) Regeneration of somatic embryos followed by their germination.
The development of an organized structure, like root, shoot or somatic embryo from cultured cells can be described as regeneration.
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1. Shoot Regeneration:
Shoot regeneration is promoted by a cytokinin like BAP. However, root regeneration is promoted by an auxin like NAA (naphthalene acetic acid). The shoot and root regeneration are generally controlled by auxin-cytokinin balance. Usually, an excess of auxin promotes root regeneration, whereas that of cytokinin promotes shoot regeneration. Roots regenerate from the lower end of these shoots to give complete plantlets.
2. Somatic Embryo Regeneration:
A somatic embryo develops from a somatic cell. The pattern of development of a somatic embryo is comparable to that of a zygotic embryo. Somatic embryo regeneration is induced by a high concentration of an auxin, such as 2, 4-D. These embryos develop into mature embryos. Mature somatic embryos or embryoids germinate to give complete plantlets.
Establishment in the Field:
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The plantlets are removed from culture vessels and established in the field. This transfer is done by specific procedures called hardening. During hardening, plantlets are kept under reduced light and high humidity Hardening procedures make the plantlets capable of tolerating the relatively harsher environment outside the culture vessels (Figs. 1.9 & 1.10).
Both callus and suspension cultures have a number of uses as:
1. Synthesis of important biochemical by forming a large cell biomass.
2. Rapid cloning and formation of plants.
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3. Genetic engineering and formation of transgenic plants.
4. Somatic hybridization.
5. Development of rapid mutations
6. Development of resistance to various stresses.
7. Induction of resistance to weedicides.
