Tissue Culture Techniques; Process; Setup Loan

Tissue Culture Techniques

Tissue Culture Techniques in Plants, Procedure of Tissue Culture and Tissue Culture Developement Setup Loan.

Introduction:

What is tissue culture? Tissue culture is the cultivation of a plant during the use of a cutting or other plant tissue. Tissue culturing is the most common form of plant reproduction & cloning in the greenhouse setting. Tissue culture can be used to reproduce plants that demonstrate exceptional disease resistance, or some other desired characteristic. The cultured tissue may consist of a single cell, a population of cells, and a part of an organ. Cells in culture may multiply; change size, form, exhibit specialized activity; or interact with other cells.

Tissue culture techniques are often used for the commercial making of plants as well as for plant research. Tissue culture involves the utilization of small pieces of plant tissue, which are cultured in a nutrient medium under sterile conditions. Using the suitable growing conditions for each explants type, plants can be induced to rapidly create new shoots, and, with the addition of proper hormones new roots. These plantlets can be divided, to produce large numbers of new plantlets. The new plants can then be placed in soil & grown in the normal manner. Tissue culture media, it is a group of systems used to grow plant cells, tissues or organs under sterile conditions on a nutrient culture medium of the known composition. Plant tissue culture is widely used to generate clones of a plant in a method known as micropropagation.

Importance of tissue culture:

• In a relatively short time & space, a large number of plantlets can be produced starting from the single explant.
• Taking an explant does not generally destroy the mother plant, so rare and endangered plants can be cloned safely.
• It is easy to select desirable traits directly from the culture setup (in vitro) thereby decreasing the amount of space necessary, for field trials.
• Once established, a tissue culture line can give a continuous supply of young plants throughout the year.
• The time required is much shortened, no want to wait for the whole life cycle of seed development. For species that have long generation times, low stage of seed production, or seeds that readily do not germinate, rapid propagation are possible.
• In vitro growing plants usually free, from the bacterial &  fungal diseases.  Virus eradication & maintenance of plants in the virus-free state.  This facilitates the progress of plant across international boundaries.
• Plant tissue culture banks can be frozen & then regenerated through tissue culture. It preserves the pollen & cell collections from which plants may be propagated. 

Types of Tissue Culture:

Seed Culture

Seed culture is the kind of tissue culture that is primarily used for plants such as orchids. For this method, explants are obtained from an in-vitro derived plant & introduced into an artificial environment, where they get to proliferate.

Embryo Culture

Embryo culture is the type of tissue culture that involves the isolation of an embryo from a known organism for in vitro growth. Embryo culture may occupy the use of a mature or immature embryo. Whereas mature embryos in culture are mainly obtained from ripe seeds, involves the use of immature embryos from hybrid seeds that failed to germinate. In doing so, the embryo is ultimately able to create a viable plant. For embryo culture, the ovule, seed from which the embryo is to be obtained is sterilized, and the embryo does not have to be sterilized again. Salt sucrose may be used to give the embryo with nutrients. The embryo culture is enriched with organic or inorganic compounds, inorganic salts as well as growth regulators.

Callus culture

Callus culture is formed by the proliferation of the parent tissue. The cells of a callus are parenchymatous, amorphous & unorganized. Generally, callus culture is formed as a result of an injury at the cut ends of a stem or a root. A localized center of activity is re­corded in a callus.

Organ culture:

That may allow differentiation & preservation of the architecture. The organ culture refers to the in vitro culture & maintenance of an excised organ primordial or whole or part of an organ in the way and function.

Anther culture

Androgenesis is the in vitro development of haploid plants originating from potent pollen grains through a series of cell division & differentiation.

Somatic Embryogenesis

This is the process of a single or group of cells initiating the growth pathway that leads to the reproducible regeneration of monozygotic embryos capable of germinating to form complete plants.

Processing Of Cultured Cells and Tissues:

Live cultures may be examined directly with a microscope, or they may be observed by means of photographs & motion pictures taken through the microscope. Cells, tissues, and organs may be killed, fixed, and stained for further examination. Following fixation, samples can be embedded and cut into thin sections to disclose additional details under a light or electron microscope.

Cells in plant tissue culture are subjected to a broad range of experimental treatment. For example, viruses, drugs, vitamins, disease-causing microorganisms may be added to the culture. Scientists then observe the cells, looking for global changes in cell behavior for changes in specific molecules, such as alterations in the expression of a particular protein.

Biological Insights:

Plant tissue culture has enabled numerous discoveries in the biological sciences. It has revealed, basic information about cells regarding their composition and form; their biochemical, genetic, and reproductive activity; and their nutrition, metabolism, specialized functions. It is also processes of aging and healing; the effects on cells of physical, chemical, and biological agents; and the differences between normal cells and abnormal cells, such as cancer cells. Work with plant tissue cultures has helped to recognize infections, and chromosomal abnormalities, to categorize tumors, and to formulate & test drugs and vaccines.

Since the discovery that certain viruses grow in tissue culture, the technique has been used to produce vaccines against poliomyelitis, measles, mumps, and other infectious diseases. Cell cultures have produced viral inhibitors, including interferon. Hormones are produced from cultures of cells or organs. Cultured white blood cells from two individuals can be used to determine compatibility between potential donors & recipients of tissue transplants.

Major Steps of Plant Tissue Culture:

Initiation Phase:

The initiation phase is the first part of plant tissue culture. Here, the tissue of interest is obtained and sterilized in order to prevent any microorganism from negatively affecting the process. It is during this phase that the tissue is initiated into the culture.

Multiplication Phase:

The multiplication phase is the second step of tissue culture where the in vitro plant material is re-divided & then introduced into the medium. Here, the medium is composed of appropriate components for growth, including regulators & nutrients. These are responsible for the proliferation of the tissue and the creation of multiple shoots. 

Elongation:

Plants are transferred again, in the laminar flow hood, onto a medium that makes the shoots elongate. This is the stage where the stems of the plants grow in length & begin to resemble little plants.

Root formation:

It is at this stage that roots are formed. Here, hormones are required in order to induce rooting, & consequently complete plantlets. This can occur in the tissue culture labor in the greenhouse, depending on the species. The elongated shoots are trimmed off the base tissue & placed onto media that contain auxins, for rooting. Once roots are visible, the plants can be planted into the greenhouse. Plants coming from the lab environment have been growing at high relative humidities are very tender. They have to be looked at very closely when they are moved to the greenhouse to make sure they are acclimatizing properly.

Equipment used for tissue culture:

Propagation by tissue culture needs a number of laboratory equipment. The various equipment and their functions are;

Autoclave: Sterilization of all glass apparatus & culture media can be accomplished by means of steam generated in the autoclave.

Analytical or Top Pan Balances: For accurate measurement of different constituents of culture media, these balances would be required. Top pan balance is used for measuring bigger quantities, while the analytical balance is used for measuring smaller quantities.

pH meter: It is used for measuring & adjusting hydrogen ion concentration of the culture media or solution. Hydrogen ion concentration wants to be maintained accurately for achieving optimum growth of plants.

Laminar Air-flow cabinets: In these cabinets shoots developed on explants are separated from clusters & transferred to fresh medium under sterile condition.

Distillation sets: Water to be used for the preparation of culture media must be free from all impurities and salts.

Air Conditioners with Stabilizers: Maintenance of preferred temperature in growth room, inoculation room or culture transfer room would be possible with air-conditioning these areas.

Microscopes: a) Stereo Microscope: This would permit dissecting out small size meristem from shoot tips by removing the protective covers of leaf primordia.

1. b) Compound microscope: This enables detection of bacteria & fungi in culture and plant tissues.

Bottle Washing Unit: Since a large number of bottles or vessels in which plants will be developed are required to be washed repeatedly before use, an automatic bottle-washing unit would be helpful.

Trays: Supporting structure for culture bottles or vessels.

Hatches: Pass through boxes used as a gateway between clean area & semi-clean area for exchanging materials.

Dissecting Kits: These are necessary for separation of shoots & preparation of micro cuttings.

Uses of Plant tissue culture:

Plant tissue culture is used to increase thousands of genetically identical plants from one single parent plant called soma clones. Since this micropropagation process produces new plantlets by the score of thousands, it has been used widely for the making of commercially important plants including food plants like tomato, banana, and apple, etc. An example of the application of micropropagation was observed in the farming of orchids as it rose exponentially due to the accessibility of millions of plantlets thanks to tissue culture methods. Why is plant tissue culture important? In this culture, the growth medium or culture solution is very important as it is used for rising plant tissue because it contains various plant nutrients in the form of ‘jelly’ known as agar & plant hormones which are necessary for the growth of plants.

Requirements of Plant Tissue Culture:

Scope and Training of Plant Tissue Culture, it is referred to the in vitro cultivation of plants, seeds and various parts of the plants (organs, embryos, tissues, single cells, and protoplasts).

Plant tissue culture is one of the most rapidly growing areas of biotechnology because of its high potential to develop improved crops & ornamental plants. With the advances made in the tissue culture, technology, it is now probable to regenerate species of any plant in the laboratory. To get the target of creating a new plant or a plant with desired characteristics, tissue culture is often coupled with recombinant DNA technology. The techniques of plant tissue culture have largely helped in the green revolution by improving the crop yield & quality. The information obtained from plant tissue cultures has contributed to our understanding of metabolism, growth, differentiation & morphogenesis of plant cells. Because of the wide range of applications, plant tissue culture attracts the attention of molecular biologists, plant breeders & industrialists. Who introduced plant tissue culture? In the 19th century, the idea of experimenting with tissue and organs of plants under controlled laboratory conditions was born. For the first time in 1902, a German physiologist, Gottlieb Haberlandt, developed the theory of in vitro cell culture.

In line with the technology & objective of tissue culture propagation, various facilities may also be required below;

Land: It is required to set up a laboratory, mother plant unit, greenhouse & office. Space may be required for installing tube well or dug well and parking of vehicles.

Mother Plants: Mother Plants would provide a source of tissues. Their performance must be tested before use, as a source of explants. In case of a tie-up with well-established laboratories, explants from testing mother plants can be available free of cost. Otherwise, collection, maintenance & testing of superior mother plants would be necessary.

Laboratory: A tissue-culture laboratory normally comprises of media preparation room, media storage room, inoculation room, culture transfer room, washing area, etc. The floor plan must be designed to promote maximum efficiency. The design must facilitate maintenance of optimum temperature, humidity, illumination & ventilation. Correct design of a laboratory will not only reduce contamination but achieve high efficiency in work performance. Properly planned & designed laboratories can reduce both the operational and energy costs.

Greenhouse in Tissue culture: In tissue-culture propagation, a greenhouse may be required to raise & maintain mother plants so that growth of organs suitable for tissue culture is maximum particularly in case of ornamentals and to harden the plantlets regularly in a natural environment. The greenhouse will enable the control over light intensity and humidity, which is essential for hardening of plants.

Electricity: As would be evident from the preceding paragraphs, no tissue-culture laboratory can function without electricity. Electricity is essential to provide the required intensity of light to the growing tissues & shoots while they are in the laboratory, to operate various equipment & facilities, which include air conditioners.

Water: Water for growing mother plants, hardening of plantlets, canteen, toilets, etc. Distilled water is required for the preparation of culture media & reagents.

Raw materials: Raw materials required for the tissue-culture project, apart from explant, are different constituents of culture media.

Skilled Manpower: Plant tissue culture is a highly skilled operation. It would, therefore, be necessary that laboratory and greenhouse workers are well qualified & experienced in the technology. Their training in well-established commercial laboratories would be useful.

Plant tissue culture stages:

Which plants are grown by tissue culture? The new plants can then be placed in soil & grown in the normal manner. Many types of plants are appropriate for use in the classroom. Cauliflower, rose cuttings, African violet leaves & carnation stems will all easily produce clones through tissue culture. 

Preparation of nutrient medium

A semi-solid medium is prepared in double distilled water containing macro elements, microelements, amino acids, vitamins, iron source, carbon source like sucrose & phytohormones. The medium is heated for dissolving the agar and 25 ml to 50 ml is dispensed into each wide mouth bottles. The vessels containing culture media are then sealed & sterilized by autoclaving.

Establishment of aseptic culture

The starting material for the procedure is generally an actively growing shoot tip of auxiliary or terminal bud or shoot tip of a plant. The procedure of plant tissue culture starts from the selection of mother plants having the desired characteristics.

Ex-plant if possible the meristematic tissue of the selected mother plant is isolated. The excised tissue/explant is washed with water & then rinsed with a disinfectant such as savlon or Dettol solution followed by a sterile-water wash. The tissue is then dipped in 10% bleach solution for ten minutes for disinfecting the plant tissue material, killing most of the fungal & bacterial organisms. Sterilization procedure of explants depends on the plant species and types of explants

Inoculation

Inoculation process is carried out under aseptic conditions. In this procedure explants or micro shoots are transferred onto the sterilized nutrient medium.

Development of plants in the growth room

After the inoculation of the plant tissue, the bottles are sealed & transferred into the growth room to trigger a developmental process under diffused light at 23 to 27°C and 50 to 60% relative humidity. Light and temperature requirements vary from species to species & sometimes during the various stages of developments.

The cultures are observed daily for growth and any signs of infection or contamination. Cultures, that do not show excellent growth or infected, are discarded. The healthy cultures develop into small shoot buds. These are subcultured on the fresh medium after four weeks. The number of subcultures necessary is specific to the plant species, which are standardized. The shoots generally develop after four weeks. After a sufficient number of shoots is developed in each container, to a minimum height of 2 cm they are transferred to another medium for initiating the procedure of rooting. The constituent of rooting standard for each plant species is specific. Roots are generally formed within two to four weeks. Plants at this stage are delicate & require careful handling.

Hardening of micro plants

Due to very high humidity inside the culture vessel & artificial conditions of development, the plantlets are tender and are therefore not ready for coping up with the field conditions. The plants removed from the sterile medium are washed & are maintained under intermittent mist or are covered with clean transparent plastic. After 10 to 15 days under high humidity, the plants are transferred to greenhouse & maintained for another 4 to 6 weeks. They are then complete to be transferred to a new house or the field. Normally, the plant tissue culture plants are sold either as ex-agar plants or hardened plants from the greenhouse.

The procedure of Plant tissue culture:

The parts of the plant used for culturing is called as explants. The explants are cultured in-vitro on a nutrient medium that caters to complete its nutritional requirements. The nutrient medium must give the following:-

1. Macronutrients – This includes elements like nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), sulfur (S) which is necessary for proper growth & morphogenesis.
2. Micronutrients – Elements like iron (Fe), manganese (Mn), zinc (Zn), etc., which is crucial to the growth of tissues.
3. Carbon or Energy source – This is one of the main crucial ingredients of the nutrient media. Sucrose is the widely used carbon source among other carbohydrates.
4. Vitamins, amino acids, & other inorganic salts.

The culture media also serve as a medium for supplying plant growth regulators to the issues which bring about their morphogenesis as per requirement. The tissues of the explants first lose their specificity to form a hard brown lump called callus. The callus then splits to increase a plant organ or a whole new plant depending upon the quantity & composition of phytohormones supplied. The entire procedure requires strict aseptic conditions to be maintained at all times as single contamination can ruin a whole batch of plants. Why tissue culture is called micropropagation in vitro? Tissue culture is a propagation method used to generate plants under sterile conditions. It is called as in vitro micropropagation, because, the explants are grown on medium & the genetic resources are conserved in the form of. Micro propagated plants are free from viruses and show increased productivity & yield.

Advantages of Plant Tissue Culture:

Mass multiplication of elite clones: Micropropagation allows the creation of large numbers of plants from small pieces of the mother plant. The production requires moderately short periods of time to grow plants. Depending on the species under production, one ex-plant can be multiplied into several thousand plants in less than one year.

Plant improvement through tissue culture: Creation of superior varieties of agricultural crops is possible through plant tissue culture method, which otherwise is not possible during conventional plant breeding methods.

True to Type production: a Large number of true to the type plants can be propagated within a short time and space and that too throughout the year. For example, it may be possible to propagate 2 to 4 lakhs of tissue cultured plants from a single bush or rose against 10 to 15 plants by conventional means. Also, it may take about 2 to 4 months to produce a healthy planting material by tissue culture means, whereas a minimum of 6 to 8 months is required for most species by the latest method of plant propagation.

Higher yields: Tissue Culture Plants may have increased branching & flowering, greater vigor and higher yield, mostly due to the possibility of elimination of diseases.

Beneficial when conventional propagation is difficult: The process may succeed to propagate plants where seeds or conventional propagation is not possible or undesirable.

Flexible method: The flexibility of nurseries can be better. As the capital investment on the mother plant is reduced to almost zero, it can be easier to adapt to changing conditions.

The innovation of new varieties: Plant tissue culture can be utilized for breeding new varieties.

Financial assistance in Tissue culture:

The tissue-culture export-oriented developments are eligible for refinance support by NABARD. Banks can provide loans for the activity provided the scheme is technically feasible & financially viable.

Bank Loan:

A bank loan of 75 to 80 % of the total cost of development shall be available from the financing institution. Bank loan considered in the model is 75%.

The rate of interest:

Banks are free to choose the rate of interest within the overall RBI guidelines issued from time to time. However, the final lending rate has been considered as 12% for working out the bankability of the model project.

Last Updated: March 30, 2019