The tissue culture method is one of the main tools of modern biotechnology, allowing to solve practical problems of physiology, biochemistry and plant genetics. Artificial cultivation of the material is carried out in compliance with certain conditions: sterilization, temperature and exposure to a special nutrient medium.
Essence
The tissue culture method is their long-term preservation and / or artificial cultivation in laboratory conditions on a nutrient medium. This technology allows you to create a biological model for studying various processes in cells that exist outside the body of plants, humans and animals.
The basis of propagation of plant tissue culture is the property of totipotency - the ability of cells to develop to the whole organism. In animals, this is realized only in fertilized eggs (with the exception of some types of intestinal).
History of development
The first attempts to cultivate plant tissues were made by German scientists at the turn of the XIX-XX centuries. Despite the fact that they were unsuccessful, a number of ideas were formulated, which were confirmed in the future.
In 1922, V. Robbins and V. Kotte, independently of each other, were able to grow the ends of the roots of corn and tomatoes on an artificial nutrient medium. A detailed study of the technique of cell and tissue culture began in the 30s. XX century R. Gotre and F. White proved that with periodic transplantation of tissue cultures into a fresh nutrient medium, they can grow indefinitely.
By 1959, 142 plant species were already grown under laboratory conditions. In the second half of the XX century. the use of dispersed (fragmented) cells also began.
Types of test material
There are 2 main types of plant tissue cultures:
- Obtained without destruction and retaining the characteristic features inherent in a living organism.
- Recoverable by cleavage (chemical, enzymatic or mechanical) from primary tissue. May be formed from one or more cell cultures.
According to the method of cultivation, the following methods are distinguished:
- on the "nursing layer", in which a substance that stimulates tissue growth, produce dividing cells of the same plant species;
- using tissue - "nanny", which is located next to the cultured cells;
- the use of a nutrient medium from an isolated dividing cell group;
- growing individual single cells in a microdrop that is saturated in composition.
Cultivation of individual cells is associated with certain difficulties. In order to artificially “force” them to share, they must receive a signal from neighboring, actively functioning cells.
One of the main types of tissue for physiological studies is callus, which occurs when adverse external factors (usually with mechanical injury). They have the ability to lose the specific characteristics inherent in the source tissue. As a result, callus cells begin to actively divide and parts of the plant form.
The necessary conditions
The success of the tissue and cell culture method depends on the following factors:
- Compliance with sterility. For transplants, special boxes are used with the supply of purified air, equipped with ultraviolet lamps. Aseptic processing should be performed on tools and materials, clothing and hands of staff.
- The use of specially selected nutrient media containing carbon and energy sources (usually sucrose and glucose), micro and macro elements, growth regulators (auxins, cytokinins), vitamins (thiamine, riboflavin, ascorbic and pantothenic acid and others).
- Compliance with temperature (18-30 ° C), light mode and humidity (60-70%). Most callus tissue cultures are grown under diffuse light since they do not contain chloroplasts, but some plants require backlighting.
Ready-to-use commercial formulations are currently used as nutrient media (Murasige and Skoog, Gamborg and Eveleg, White, Kao and Mikhaylyuk and others).
Advantages and disadvantages
The advantages of the cell and tissue culture method are:
- good reproducibility of the results;
- regulation of intercellular interactions;
- low consumption of reagents;
- genetic homogeneity of cell lines;
- the possibility of mechanization of the growing process;
- control over cell conditions;
- low temperature storage of live cultures.
The disadvantage of this biotechnology include:
- the need to comply with strict aseptic conditions;
- the instability of the properties of cells and the possibility of their undesirable mixing;
- the high cost of chemicals;
- incomplete equivalence of cultured tissues and cells in a living organism.
Application
The tissue culture method is used to conduct research:
- processes inside the cells (synthesis of DNA, RNA and proteins, metabolism and the effect on it with the help of drugs);
- intercellular reactions (the passage of substances through cell membranes, the activity of the hormone-receptor complex, the ability of cells to stick together, the formation of histological structures);
- interactions with the environment (absorption of nutrients, infection by infections, the processes of nucleation and development of tumors, and others);
- results of genetic manipulations with cells.
Promising areas of biology and pharmacology, the development of which uses this technology, are:
- obtaining effective herbicides, growth regulators for agronomic crops, biologically active compounds for use in the manufacture of medicines (alkaloids, steroids and others);
- directed mutagenesis, elimination of new hybrids, overcoming postgamous incompatibility;
- clonal propagation, which allows you to get a large number of genetically identical plants;
- elimination of virus-resistant and virus-free plants;
- cryopreservation of the gene pool;
- tissue reconstruction, creation of stem cell sources (tissue engineering).