Tissue culture is the process of maintenance and growth of cells and tissues in suitable, aseptic, artificial nutrients medium. Plant tissue culture process involves the culture of plant cells, tissues and organs.
This process is based on the unique property of plant cells known as totipotency. Gottilieb Haberlandt (1902) who discovered totipotency is also credited for attempting to cultivate plant leaf cells in simple nutrient medium for the first time. In the recent times, plant tissue culture technique has become a major tool in crop improvement, experimental biology and also in fundamental or applied research.
Tissue culture techniques
Some basic steps are followed in plant tissue culture which is described below.
(1) Explant selection
Explant is the plant part used for tissue culture. Explant from healthy and young part of the plant is used. Parenchymas from stems, rhizomes, tubers, roots are easily accessible and respond quickly to culture condition.
Sterilization means elimination of all living organisms (microbes). The tissue culture is carried out in completely aseptic condition for which there is a need to properly sterilize all the glasswares culture media and explants. The explants are surface sterilized by repeated washing in sterile water and by using disinfectants such as mercuric chloride, hydrogen peroxide etc. The glassware, culture media and other instruments are sterilized in an instrument called autoclave where sterilization is done in steam under high pressure or in a preasure cooker.
(3) Preparation of nutrient or culture medium
The culture medium is prepared in aseptic condition. The basic constituents of any culture medium are:
Inorganic nutrients: Inorganic nutrients inculde macronutrients as salts of nitrogen, phosphorous, potassium, calcium, magnesium and sulphur and micronutrients like boron, molybdenum, copper, zinc. Iron and chloride
Source of carbon
Sucrose is used as the source of carbon.
Several growth hormones like 2,4,1), cytokinins-benzylaminopurine, kinetin, myoinositol, 1AA, XAA arc used, vitamins like nicosstinic acid, pyridoxinc-IICL are added to the medium.
After the addition of all the ingredients in appropriate proportion, agar-agar is added to prepare a solid medium. In some types of culture (like root culture) liquid medium is used (no agar-agar used).
Inoculation is the process of the transfer of explant to suitable nutrient medium contained in culture vessels. This is done in sterile condition either in an inoculation chamber or laminar flow. After the inoculation the culture vessels are maintained in controlled temperature and light. The suitable temperature, for tissue culture ranges between i8-25°c.
(5) Callus formation and its culture
A callus is an amorphous mass of loosely arranged thin walled parenchyma cells developing from proliferating cells of parent tissue (Dodos & Roberts, 1985). The nutrient medium supplemented with auxins induces cell division and soon the upper surface of explant is covered by callus.
The callus has the biological potential to develop normal root, shoots and ultimately forming a plant. Callus is formed through three developmental stages: induction, cell division and differentiation. Callus formation is governed by the source of explant, nutritional composition of the medium and environmental factors.
During the induction period the metabolic rate of cells is stimulated. Owing to increased metabolic rate cells enter to cell division stage. In the third phase, cellular differentiation and expression of certain metabolic pathways start leading to secondary products. When callus are grown on a nutrient medium for a long time, it becomes essential to subculture it in fresh media.
Organogenesis means the development of organs like root, shoot and leaves (but not embryo). Organogenesis starts with stimulation caused by the chemical of medium, substances carried over from the original explants and endogenous compounds produced by the culture.
Organogenesis can be induced with the application of varying proportions of auxin and cytokinin. Skoog and Miller (1957) demonstrated that a high ratio of auxin: cytokinin (3 : 0.02) stimulated root formation in tobacco callus but a low ratio of the same (3 : 0,2) induced shoot formation.
(7) Somatic Embryogenesis
This is the process of inducing embryo formation from somatic cells of cultured plant tissue. The embryo thus developed is knowTi as embryoids. Two different nutritional media are required to obtain embryoids.
First medium contains auxin to initiate embryogenic cells. Second medium lacks auxin or has reduced level of auxins for subsequent development of embryonic cells into embryoids and plantlets.
The embryogenic cells pass through three different stages, e.g. globular, heart shaped and torpedo shaped to form embryoids. Some plants in which somatic embryogenesis has been induced in vitro are Atropa belladona, Brassica oleracea, Carica papaya, Coffea arabica, Citrus cinensis, Daucus carroia, Nicotiana tobacum etc.
Somaclonal Variations: In 1981, P.J Larkins and W. R. Scowcrott at the Division of Plant Industry, C.S.I.R.O. Australia gave the name somaclonal variation to genetic variability generated during tissue culture.
Though the cultured tissues are grown from single explant, over a long period of maintenance genetic variabilities are marked in the cultures. This may be due to (i) reflection of heterogeneity between cells and explant tissues, (ii) a simple representation of spontaneous mutation rate or (iii) activation by culture environment of transposition of genetic materials. Somaclonal variants of leaf callus culture of Solanum tuberosum have shown characters like disease resistance, variations in maturity dates of tubers, yield and shape.
Cell suspension cultures: Cell suspension is prepared by transferring a fragment of callus (about 500 mg) to liquid medium (500 ml) and agitating them aseptically in a shaker to make the cells free. The suspension then includes single cell, cell aggregates, and residual including dead cells. A good suspension contains high proportion of single cells. Cell suspension cultures have many advantages over the callus cultures as below:
(i) The cell suspension can be pipetted.
(ii) They are less heterogeneous.
(iii) They can be cultured in volumes up to 1,500 liters.
(iv) They can be subjected to more stringent environmental controls.
(v) They can be manipulated for production of natural products.