The method of tissue culture is one of the main tools of modern biotechnology, allowing to solve practical problems of plant physiology, biochemistry and genetics. Artificial cultivation of the material is carried out subject to certain conditions: sterilization, temperature control and exposure to a special nutrient medium.
Essence
The method of tissue culture 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 reproduction of plant tissue culture is based on the property of totipotency - the ability of cells to develop into a whole organism. In animals, this is realized only in fertilized eggs (with the exception of some types of coelenterates).
Development history
The first attempts to cultivate plant tissues were made by German scientists at the turn of the 19th-20th centuries. Despite the fact that they were unsuccessful, a number of ideas were formulated, which were confirmed later.
In 1922, W. Robbins and W. Kotte, independently of each other, were able to grow the tips of corn and tomato roots on an artificial nutrient medium. A detailed study of cell and tissue culture techniques began in the 1930s. 20th century R. Gautre 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 being grown under laboratory conditions. In the second half of the XX century. the use of dispersed (separated) cells has also begun.
Types of test material
There are 2 main types of plant tissue cultures:
- Produced without destruction and preserving the characteristic features inherent in a living organism.
- Extracted by degradation (chemical, enzymatic or mechanical) from primary tissue. Can be formed from one or more cell cultures.
The following methods are distinguished by the method of cultivation:
- on the "feeding layer", in which a substance that stimulates tissue growth is secreted by dividing cells of the same plant species;
- using the nurse tissue next to the cultured cells;
- use of nutrient medium from an isolated dividing cell group;
- growing individual single cells in a microdroplet saturated in composition.
Cultivation from single cells is fraught with certain difficulties. In order to artificially "force" them to divide, they must receive a signal from neighboring, actively functioning cells.
One of the main types of tissue for physiological research is callus, which occurs under adverse external factors (usually mechanical injury). They have the ability to lose the specific characteristics inherent in the original tissue. As a result, callus cells begin to actively divide and parts of the plant are formed.
Necessary conditions
The success of the tissue and cell culture method depends on the following factors:
- Compliance with sterility. For transplantation, special boxes with purified air supplied, equipped with ultraviolet lamps, are used. Tools and materials, clothes and hands of personnel should be subjected to aseptic processing.
- The use of specially selected nutrient media containing sources of carbon and energy (usually sucrose and glucose), micro- and macronutrients, growth regulators (auxins, cytokinins), vitamins (thiamine, riboflavin, ascorbic and pantothenic acid and others).
- Compliance with temperature (18-30 ° C), light conditions and humidity (60-70%). Most callus tissue cultures are grown under ambient light as they do not contain chloroplasts, but some plants require backlighting.
Currently ready-madecommercial lineups (Murasige and Skoog, Gamborg and Eveleg, White, Kao and Mikhailyuk and others).
Pros and cons
The advantages of the cell and tissue culture method are:
- good reproducibility of the obtained results;
- regulation of intercellular interactions;
- low consumption of reagents;
- genetic homogeneity of cell lines;
- possibility of mechanization of the growing process;
- control over cage conditions;
- low temperature storage of live cultures.
The disadvantage of this biotechnology is:
- need to comply with strict asepsis conditions;
- instability of cell properties and the possibility of their undesirable mixing;
- high cost of chemicals;
- incomplete equivalence of cultured tissues and cells in a living organism.
Application
Tissue culture method used for research:
- processes inside cells (synthesis of DNA, RNA and proteins, metabolism and influence on it with the help of drugs);
- intercellular reactions (the passage of substances through cell membranes, the work of the hormone-receptor complex, the ability of cells to adhere to each other, the formation of histological structures);
- interactions with the environment (absorption of nutrients, transmission of infections, processes of origin and developmenttumors and others);
- results of genetic manipulations with cells.
Promising areas of biology and pharmacology, in the development of which this technology is used, are:
- obtaining effective herbicides, growth regulators for agronomic crops, biologically active compounds for use in the production of medicines (alkaloids, steroids and others);
- directed mutagenesis, breeding of new hybrids, overcoming postgamous incompatibility;
- clonal propagation, which allows you to get a large number of genetically identical plants;
- breeding of virus-resistant and virus-free plants;
- cryopreservation of the gene pool;
- tissue reconstruction, creation of stem cell sources (tissue engineering).
