Department of Botany

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“Biotechnology”

Presented By

Dr P Y Anasane

G S Gawande Mahavidyalaya, Umarkhed

Plant Tissue Culture:

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Tissue culture is a process that involves exposing plant tissue to a specific regimen of nutrients, hormones, and light under sterile, in vitro conditions to produce many new plants, each a clone of the original mother plant, over a very short period of time, another way of cloning plants is by tissue culture, which works not with cuttings but with tiny pieces from the parent plant. Sterile agar jelly with plant hormones and lots of nutrients is needed, cells grow rapidly into small masses of tissue.

GOTTLIEB HABERLANDT

(The father of plant tissue culture)

Different techniques in plant tissue culture may offer certain advantages over traditional methods of propagation are follows.

1. The regeneration of whole plants from plant cells that have been genetically modified.
2. The production of plants in sterile containers that allows them to be moved with greatly reduced chances of transmitting diseases, pests, and pathogens.
3. The production of exact copies of plants that produce particularly good flowers, fruits, or have other desirable traits.
4. To quickly produce mature plants.
5. The production of multiples of plants in the absence of seeds or necessary pollinators to produce seeds.
6. To clear particular plants of viral and other infections and to quickly multiply these plants as 'cleaned stock' for horticulture and agriculture.

Some Uses or Applications: Plant tissue culture is used widely in the plant sciences, forestry, and in horticulture and are following applications.

1. A plant breeder may use tissue culture to screen cells rather than plants for advantageous characters, e.g. herbicide resistance.
2. To rapidly study the molecular basis for physiological, biochemical, and reproductive mechanisms in plants, for example in vitro selection for stress tolerant plants.
3. The commercial production of plants used as potting, landscape, and florist subjects, which uses meristem and shoot culture to produce large numbers of identical individuals.
4. To conserve rare or endangered plant species.
5. Large-scale growth of plant cells in liquid culture in bioreactors for production of valuable compounds, like plant-derived secondary metabolites and recombinant proteins used as biopharmaceuticals.
6. To cross distantly related species by protoplast fusion and regeneration of the novel hybrid.

Laboratory Requirements and Infrastructure

‘Plant tissue culture’ or in vitro cultivation of plant parts needs some basic requirements:

1. Cultivation should be done under aseptic conditions.
2. The isolated plant part should get an appropriate environment which will help to divide the cell and to get an expression of internal potential.

Basic facilities for plant tissue culture operations involving any type of in vitro proce­dures must include certain essential elements:

1. Washing and storage facilities;
2. Media preparation, sterilization and storage room;
3. Transfer area for aseptic manipulations;
4. Culture rooms or incubators for maintenance of cultures under controlled condi­tions of temperature, light and humidity;
5. Observation or data collection area;
6. Transplantation area.

Washing and Storage Facilities : An area with large sink (lead lined to resist acids and alkalis) and draining area is necessary with provision for running water, draining-boards or racks and ready access to a deionized, distilled and double-distilled apparatus. Space should also be available to set up drying ovens, washing machines, plastic or steel buckets for soaking labware, acid or deter­gent baths, pipette washers, driers and cleaning brushes. For storage of washed and dried labware, the laboratory should be provided with dustproof cupboards or storage cabinets.

Media Preparation Room or Space : This part is the central section of the laboratory where most of the activities are performed i.e., media preparation and sterilisation of media and glassware’s needed for culture. There should be sufficient working bench as well as storage space.

The essential in the room : Different types of glassware, balances, required chemicals, hot plates and Stirrer, water bath, pH meter, autoclave and Hot air oven, microwave oven, shaker, centrifuge machine, refrigerator and Freezer, storage cabinet (Dust-free) etc.

Transfer Area : Tissue culture techniques can only be successfully carried out in a very clean labora­tory having dry atmosphere with protection against air-borne microorganisms. For this pur­pose a sterile dust-free room/cabinet is needed for routine transfer and manipulation work.

The ‘laminar air flow cabinet’ is the most common accessory used for aseptic manipulations now-a-days. The cabinet may be designed with horizontal air flow or vertical air flow where the air is forced into the cabinet through a bacterial HEPA (High Efficiency Particulate Air) filter. The air flows over the working bench at a constant rate which prevents the particles (microorganisms) from settling on the bench. Before ope­ration in the laminar air flow cabinet, the interior of the cabinet is sterilised with the ultraviolet (UV) germicidal light and wiping the floor of cabinet with 70% alcohol. Inoculation chamber, a specially designed air tight glass chamber fitted with UV light, may also be used as transfer area.

Culture Room : Plant tissue cultures should be incubated under conditions of well-controlled tempe­rature, illumination, photoperiod, humidity and air circulation. Incubation culture rooms, commercially available incubator cabinets, large plant growth chambers and walk-in- environmental rooms satisfy these requirements. Culture rooms are constructed with proper air-conditioning; perforated shelves to support the culture vessels, fitted with fluorescent tubes having a timing device to maintain the photoperiod, black curtains may be used to maintain total darkness. For the suspension cultures, gyratory shakers are used.

Instruments :

Laminar air flow :

• A laminar flow cabinet or tissue culture hood is a carefully enclosed bench designed to prevent contamination of semiconductor wafers, biological samples, or any particle sensitive materials.
• Air is drawn through a HEPA filter and blown in a very smooth, laminar flow towards the user.
• The cabinet is usually made of stainless steel with no gaps or joints where spores might collect.
• Laminar flow cabinets may have a UV-C germicidal lamp to sterilize the interior and contents before usage to prevent contamination of experiment.
• Germicidal lamps are usually kept on for 15 minutes to sterilize the interior and no contact is to be made with a laminar flow hood during this time. During this time, scientists normally prepare other materials to maximize efficiency. (It is important to switch this light off during use, to limit exposure to skin and eyes as stray ultraviolet light emissions can cause cancer.

Laminar Air Flow or tissue culture hood

Function and Work :

• In a laminar flow hood the air is passed through a HEPA (High Efficiency Particulates Air) filter which removes all airborne contamination to maintain sterile conditions.
• Now the sterile air flows into the working (flasking) area where you can do all your flasking work without risk of contamination.
• It works by the use of in-flow laminar air drawn through one or more HEPA filters, designed to create a particle-free working environment and provide product protection.
• Air is taken through a filtration system and then exhausted across the work surface as part of the laminar flows process.

Autoclave :

• Autoclave is used to sterilize medium, glassware and tools for the purpose of plant tissue culture.
• The same equipment is used in hospitals to sterilize gauge, cotton, tools and linen, etc. Sterilization of material is carried out by increasing moist heat (121 °C) due to increased pressure inside the vessel (15-22 psi, pounds per square inch or 1.02 to 1.5 kg/cm2) for 15 minutes for routine sterilization.
• Moist heat kills the microorganism and makes the material free from microbes.

Construction :

• Autoclaves of different sizes from 5 litres to several hundred litres capacity are available in horizontal or vertical designs,
• an autoclave have a body, an internal (or external for small sized autoclaves comparable to household pressure cooker) heating system, a container to hold material, its cover fixed with pressure gauge, safety valve, pressure release valve etc.
• Lid is tightened with the help of screws and a gasket seals the body and lid. A jacket, paddle lifter, timer, and indicator etc., are also provided with large sized autoclaves. Autoclaves may be constructed of aluminum, mild steel, stainless steel or gun metal. Industrial autoclave can accommodate large trolley containing huge number of glassware’s or large bioreactors.

Autoclave

Growth chamber:

• This is called chamber where in temperature and relative humidity are controlled.
• We can maintain the temperature, relative humidity and light required for different crops. It works with same principle as that of germinator.
• This is a modified chamber of larger one and the worker can enter into it and evaluate the seedlings. Provisions are made to maintain the temperature and relative humidity.
• This is used widely in practice for Biotechnology.

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Applications of Growth chamber in Plant cell/tissue culture, germination, acclimation, or growth test Environmental test, Growth of rice, arabidopsis, etc. Insect experiment, Cell culture, environmental resistance test for plants, Storage test at constant temperature and at constant humidity.

Plant Growth Chamber.

Plant Growth Chamber is widely used in plant biology, soil science and agriculture research and growth of rice and Arabidopsis etc. The purpose of using a plant growth chamber or tissue culture growth chamber is to create artificial environment using combination of temperature, humidity and light at various ranges. Each cabinet features unmatched accuracy and reliability in control of temperature, humidity and light. In addition to standard models, large sizes / walk-in rooms (according to plant height) are also made on request.

Each growth chamber is durably constructed for long service life. The standard machines are made of powder coated mild steel from outside and stainless steel from inside. In addition, we also make plant growth chamber made of complete stainless steel 316 if needed. These units are double walled and insulated with thick 75 mm of PUF. The standard temperature range remains between 5°C to 60°C which is adjusted through digital PID controller. The humidity range remains between 50% to 90%, which is adjusted through separately fitted PID controller. The humidity system is very comfortable and have facility to auto cut-off water supply if tank is filled up to its level. Air circulation is done by axial fans. For illumination, we use detachable LED light on 3 sides of the chamber. These lights are controlled through digital timer with 24 x 7 weekly settings.

Culture Media (MS Media) : Read this article to learn about the plant tissue culture media and its types, constituents, preparation and selection of a suitable medium. Culture media are largely responsible for the in vitro growth and morphogenesis of plant tissues. The success of the plant tissue culture depends on the choice of the nutrient medium. In fact, the cells of most plant cells can be grown in culture media Basically, the plant tissue culture media should contain the same nutrients as required by the whole plant. It may be noted that plants in nature can synthesize their own food material. However, plants growing in vitro are mainly heterotrophic i.e. they cannot synthesize their own food.

Major Types of Media:

White’s medium: This is one of the earliest plant tissue culture media developed for root culture.

MS medium: Murashige and Skoog (MS) originally formulated a medium to induce organogenesis, and regeneration of plants in cultured tissues. These days, MS medium is widely used for many types of culture systems.

B5 medium: Developed by Gamborg, B5 medium was originally designed for cell suspension and callus cultures. At present with certain modifications, this medium is used for protoplast culture.

MS Media: Murashige and Skoog medium (or MSO or MS0 (MS-zero)) is a plant growth medium used in the laboratories for cultivation of plant cell culture. MSO was invented by plant scientists Toshio Murashige and Folke K. Skoog in 1962 during Murashige's search for a new plant growth regulator.

Constituents of Media : Many elements are needed for plant nutrition and their physiological functions. Thus, these elements have to be supplied in the culture medium to support adequate growth of cultures in vitro.

The culture media usually contain the following constituents:

1. Inorganic nutrients
2. Carbon and energy sources
3. Organic supplements
4. Growth regulators
5. Solidifying agents
6. pH of medium

Organic Supplements : The organic supplements include vitamins, amino acids, organic acids, organic extracts, activated charcoal and antibiotics.

Vitamins : Plant cells and tissues in culture (like the natural plants) are capable of synthesizing vitamins but in suboptimal quantities, inadequate to support growth. Therefore the medium should be supplemented with vitamins to achieve good growth of cells. The vitamins added to the media include thiamine, riboflavin, niacin, pyridoxine, folic acid, pantothenic acid, biotin, ascorbic acid, myo­inositol, Para amino benzoic acid and vitamin E.

Amino acids : Although the cultured plant cells can synthesize amino acids to a certain extent, media supplemented with amino acids stimulate cell growth and help in establishment of cells lines. Further, organic nitrogen (in the form of amino acids such as L-glutamine, L-asparagine, L- arginine, L-cysteine) is more readily taken up than inorganic nitrogen by the plant cells.

Organic acids : Addition of Krebs cycle intermediates such as citrate, malate, succinate or fumarate allow the growth of plant cells. Pyruvate also enhances the growth of cultured cells.

Organic extracts : It has been a practice to supplement culture media with organic extracts such as yeast, casein hydrolysate, coconut milk, orange juice, tomato juice and potato extract.

Antibiotics : It is sometimes necessary to add antibiotics to the medium to prevent the growth of microorganisms.  

Growth Harmon’s : Three types of plant hormones are usually recognized. These are auxins, gibberellins and cytokinins. These were discovered in the early decades of the twentieth century, in 1930’s and in 1960's respectively. Naturally, the knowledge accumulated on auxins and gibberellins is far greater than that gathered for cytokinins. Plant hormones or phytohormones are a group of natural organic compounds 

Auxins : Kogl and Haagen-Smit (1931) introduced the term ‘auxin’ (auxeinG = to grow or to increase) for designating those plant hormones which are specially concerned with cell enlargement or the growth of the the shoots.

Cytokinins : Skoog, Strong and Miller (1965) have defined cytokinins as chemicals which, regardless of their activities, promote cytokinesis (cell division) in cells of various plant organs.

Gibberellins : Gibberellins and allied substances have been found in higher plants also by Mitchell et al (1951), West and Phinney (1957) and Sumiki and Kawarada (1961).

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Sterilization Techniques : Physical Methods of Sterilization: Sun-Light: Ultraviolet rays present in the sun-light are responsible for spontaneous sterilization in natural conditions. In tropical countries the sun light is more effective in killing bacteria due to combination of ultraviolet rays and heat. By killing bacteria in suspended water, sunlight provides natural method of disinfection of water bodies such as tanks and lakes.

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Sterilization Techniques

Hot Air Oven: Articles are exposed to high temperature (160°C) for duration of one hour in an electrically heated oven (method was introduced by Louis Pasteur).

Certain other methods of sterilization at below 60°C temperature are : Vaccine bath (contaminating bacteria in a vaccine preparation can be inactivated by heating in a water bath at 60°C for one hour),  

Autoclave : Sterilization can be effectively achieved at a temperature above 100°C using an autoclave.

Chemical Methods of Sterilization : Chemicals destroy pathogenic bacteria from inanimate surfaces and are all also called disinfectants. Alcohols: E.g., Ethyl alcohol, Isopropyl alcohol and methyl alcohol. (A 70% solution kills bacteria).

Sterilization of instruments and glassware : All the glassware or plastic ware that is used for dispensing or handling the media is sterilized by autoclaving.

Tissue Culture Technique

Cellular totipotency : Totipotency is the genetic potential of a plant cell to produce the entire plant. In other words, totipotency is the cell characteristic in which the potential for forming all the cell types in the adult organism is retained.

Expression of Totipotency in Culture : The basis of tissue culture is to grow large number of cells in a sterile controlled environ­ment. The cells are obtained from stem, root or other plant parts and are allowed to grow in culture medium containing mineral nutrients, vi­tamins and hormones to encourage cell division and growth. As a result, the cells in culture will produce an unorganised proliferative mass of cells which is known as callus tissue.

Importance of Totipotency in Plant Science : The ultimate objective in plant protoplast, cell and tissue culture is the reconstruction of plants from the totipotent cell. Although the process of differentiation is still mysterious in general, the expression of totipotent cell in cul­ture has provided a lot of information’s.

Differentiation and morphogenesis : Morphogenetic responses may be induced in organisms by hormones, by environmental chemicals ranging from substances produced by other organisms to toxic chemicals or radionuclides released as pollutants, and other plants, or by mechanical stresses induced by spatial patterning of the cells. In some plant species, the crown gall bacterium (Agrobacterium tumefaciens) induces a special type of tumour, called teratomas, the cells of which possess the capac­ity to differentiate shoot buds and leaves when they are grown in culture for unlimited periods.

Morphogenesis (from the Greek morphe means shape and genesis creation, literally, "beginning of the shape") is the biological process that causes an organism to develop its shape. It is one of three fundamental aspects of developmental biologyalong with the control of cell growth and cellular differentiation, unified in evolutionary developmental biology.

Cell Differentiation : The process by which cells becomes specialized in form and function. The cells undergo change that organize them into tissues and organs.

Morphogenesis : As the dividing cells begin to take from, they are undergoing morphogenesis which ,mans the “Creation of form.” Morphogenetic events lay out the development very early on development as cell division, cell differentiation and morphogenesis overlap.

Callus Culture : Explants, when cultured on the appropriate medium, usually with both an auxin and a cytokinin, can give rise to an unorganized, grow­ing, and dividing mass of cells. This is called as callus. It is thought that any plant tissue can be used as an ex-plant, if the correct conditions are found. In culture, this proliferation can be maintained more or less indefinitely, provided that the callus is sub-cultured on to fresh medium periodically. During callus for­mation, there is some degree of dedifferentiation

Callus Culture

Explants for Callus Culture : It can be any part of the plant (root, stem, leaf, anther, flower, etc.). The plant part chosen as explants must be young and most preferen­tially meristematic in nature.

Factors Affecting Callus Culture:

1. Light requirement varies from plant to plant.
2. Temperature in the range of 22 to 28 degree centigrade.
3. Auxine and cytokinin hormones.

Applications of Callus Cultures:

1. To know the process of cell and organ dif­ferentiation.
2. Used as a study source to learn the nutri­tional requirements of plants.
3. To learn about somaclonal variation.
4. For the production of secondary metabo­lites.

Steps in Micropropogation of Shoot tip.

Technique of Micro propagation :

Micro propagation is a complicated process and mainly involves 3 stages (I, II and III). Some authors add two more stages (stage 0 and IV) for more comprehensive representation of micro- propagation.

Stage 0: This is the initial step in micro- propagation, and involves the selection and growth of stock plants for about 3 months under controlled conditions.

Stage I : In this stage, the initiation and establishment of culture in a suitable medium is achieved. Selection of appropriate explants is important.

Answer in one sentence

1. What is totipotency?
2. Plant growth chamber provided control condition of
3. What is the diameter of HEPA filter?
4. What is inoculation?
5. What is the role of agar in media?
6. What are the major inorganic nutrients (macronutreints) of media?
7. What is callus?
8. What are the minor inorganic nutrients (micronutreints) of medi media ?
9. What is the role of cytokinin in media?
10. What is the role ofauxin in media?

Write short note on

1. Laminar air flow
2. Infrastructure of plant tissue culture
3. MS media
4. Axillary bud culture
5. Differentiation
6. Totipotency
7. Morphogenesis
8. Callus culture
9. Meristem culture
10. Autoclave
11. Growth chamber
12. Growth Hormone
13. Hardening of the plant

Long answer question

1. What is totipotency? Explain the callus culture.
2. Write detailed account of MS media and add a note on growth hormone.
3. Write an account of micropropagation. Give its significance.
4. What is sterilization? Explain various techniques of sterilization.
5. Explain in detail differentiation and morphogenesis.

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