TISSUE CULTURE - 2

                                               Callus Culture

Callus is basically a more or less non-organized tumor tissue which usually arises n wounds of differentiated tissues and organs. Thus, in principle, it is a non-organized and little-differentiated tissue. The cells in callus are of
a parenchymatous nature. When critically examined, callus culture is not homogeneous mass of cells, because it is usually made up of two types of tissue: differentiated and non- differentiated. Explant tissue is a differentiated tissue (roots, stem, leaves, flowers,etc.) which is used as a starting material for callus induction. These explant tissues generally show distinct planes of cell division, cell proliferation and organization into specialized structures such as vascular systems. If there are only differentiated cells present in an isolated explant, then dedifferentiation must take place before II division can occur. Parenchyma cells present in the explant usually undergo is differentiation. If the explant already contains meristematic tissue when isolated, then this can divide immediately without dedifferentiation taking place.

Callus formation takes place under the influence of exogenously supplied growth regulators present in the nutrient medium. The type of growth regulator requirement and its concentration in the medium depends strongly on the genotype and endogenous hormone content of an explant. These requirements can be put into three categories:

1. Auxin alone (especially in monocotyledons) .

2. Cytokinin alone

3. Both auxin and cytokinin (carrot)

If the callus is difficult to induce, or if juvenile callus is needed, then immature embryos or seedlings or parts of these are used. It should be taken into account that type of starting material (juvenile or adult) and the original position of the explant in the plant reflects the endogenous hormone level which have an important influence on processes such as cell division and organ and embryo formation.

Many other factors are important for callus formation: genotype, composition the nutrient medium, physical growth factors (light, temperature, etc.). The Murashige and Skoog (1962) mineral medium, or modifications of this are often used.

Sucrose or glucose (2-4%) is usually employed as the sugar source. The effect ight on callus formation is dependent on the plant species; light may be required in some cases and darkness in other cases. A temperature of 22-28°C is normally advantageous for callus formation.

After callus induction, the callus is grown further on a new medium which referred to as subculturing. When subcultured regularly on agar medium, callus cultures will exhibit an S-shaped or sigmoid pattern of growth during each passage. There are five phases of callus growth:

1. Lag phase, where cells prepare to divide.

2. Exponential phase, where the rate of cell division is highest.

3. Linear phase, where cell division slows but the rate of cell expansion increases.

4. Deceleration phase, where the rates of cell division and elongation decreases.

5. Stationary phase, where the number and size of cells remain constant.

Callus growth can be monitored by fresh weight measurements, which convenient for observing the growth of cultures over time in a non-destructive manner. Dry weight measurements are more accurate than fresh weight, but method requires sacrifice of the samples. Mitotic index measurement of cell division rates require extensive sampling to reduce sample error and are not easy to perform.

Organ Culture

It is an isolated organ grown in vitro. It can be given different names depends upon the organ used as an explant. For example, meristem or shoot tip culture ,root culture, nucellus culture, endosperm culture, ovule culture, a culture for production of androgenic haploids while ovule and ovary culture vitro production of gynogenic haploids . The culture of plant o results in three types of in vitro culture (De Fossard et al. 1977).

Organized: The culture of whole plants (embryos, seeds) and organ has been termed as organized culture. In this, characteristic organized structure of a plant o individual organ is maintained. If the organized structure is not broken down, progeny arise which are identical to the original plant material (e.g. meristem cuIture).

Non-organized: If cells and/or tissues are isolated from an organized part of a plant, de-differentiate and are then cultured, a non-organized growth in the form callus tissue results. If the callus disperses into clumps of cells (aggregates) a single cells results, it is referred to as suspension culture. Non-organized culture have very low genetic stability.

Non-organized/organized: This type of culture is intermediate between the above two types. Cells in an isolated organ or tissue, first dedifferentiate and then form tissue which then redifferentiate to form organs (roots or shoots) or embryos. Thus organized structures can develop from non-organized cultures either through techniques or spontaneously. In this the progeny are often not completely identical to the original plant material.

Protoplast culture:

Now we will look at the different procedures of protoplast culture. They are as follows:

1. Isolation of protoplasts: Protoplasts (cell without cell wall) are the biologically active and most significant material of cell. Cooking for the first time isolated protoplasts of plant tissue by using cell wall degrading enzymes viz. cellulase, hemicellulase, pectinase, and protease extracted from a saprophytic fungus Trichoderma viride .Now the protoplasts are cultured in vitro. The basic technique s of isolation are given in Fig .

Sterilisation of the leaf sampleswith sodium hypochlorite solution.

Rinsing in suitable Osmaticum ie in distilled water or MS medium adjusted to a suitable pH and buffer to maintain osmotic pressure.

Plasmolysis of cells takes place by keeping the stripped leaves in 13% mannitol for 3 hours.

Peeled leaves are transferred into an already sterilized enzyme solution for 12-15 hours for the facilitation of the enzyme to enter the tissue.

Isolation and purification of protoplasts takes place by filtering the enzyme solution containing protoplasts through a nylon mesh (45μm).The filtrate is centrifuged 2-3 times repeating the above steps and finally a specifc concentration of protoplast suspension is prepared.

2) Protoplast culture and regeneration:

From the protoplast solution of known density (about 105protoplasts /ml) about 1ml suspension is poured on sterile and cooled down nutrient medium in Petri dishes. The plates are incubated at25^o C in a dim white light.

The protoplast regenerates a cell wall, undergo cell division and forms callus.

The callus can be subcultured. Embryogenesis begins from callus when it is placed on nutrient medium lacking mannitol and auxin.

the embryo develops into the seedling s and finally into mature plants.

Methods of isolation of protoplasts

Protoplast suspension Filter centrifuge Protoplast residue wash and decant 3 times

Plantlets Embryogenesis Protoplast regeneration Culture

  

Anther Culture :

Anther culture is the process of using anthers to culture haploid plantlets.

Microspores (premitotic stage)

Microspores(mitotic )

The technique was discovered in 1964 by Guha and Maheshwari. This technique can be used in over 200 species, including tomato, rice, tobacco, barley, and geranium. Some of the advantages which make this a valuable method for obtaining haploid plants are:

the technique is fairly simple it is easy to induce cell division in the immature pollen cells in some species a large proportion of the anthers used in culture respond (induction frequency is high) haploids can be produced in large numbers very quickly.

In experiments using Datura innoxia, induction frequencies of almost 100% and a yield of more than one thousand plantlets or calluses have occurred under optimal conditions from one anther. Success can be determined within 24 hours as cells begin to divide.

Method of anther culture

Plant Tissue Culture: Methods

What all do you require for a proper tissue culture laboratory?

The setting up of a tissue culture laboratory needs proper planning. It depends upon space availability, volume of work to be carried out and funds. Usually, the available laboratory space, is be divided into five distinct laboratory areas. These are: Media preparation area/room, Aseptic transfer chamber area, Environmentally controlled culture room, Analytical room and Acclimatisation room.

Media Preparation: Area/Room

Laboratory tables or benches and revolving stools with adjustable height, Hot plate and magnetic stirrers, analytical loading single pan balance-with precision of 0.001 g, weighing range 0.1 mg- 80g, digital read out, Top pan loading balance for quick weighing, range 100 mg-500g capacity, sensitivity 0.1 g, Refrigerator and freezer, Water purification and storage system, Glassware washing facility with proper drainage, Gas outlet, Electric hot-air oven range upto 250 °C and microwave oven, Digital pH meter, Range 0-14 pH, accuracy 0.1 pH, temperature compensation 0- 100°C, Autoclave preferably horizontal., Continuous supply of single and double distilled water.

Storing glasswares, plastic wares, chemicals, plugs and appliances required for media preparation are as follows:

Requirements: Culture tubes/conical flasks/petri dishes of various capacities, Measuring cylinders- 25 ml, 100 ml, 500 ml, 1000 mI, Cotton for plugs/plastic caps (autoclavable),

General glasses/plastic wares of various capacities such as volumetric flasks, beakers, reagent bottles, pipittes, vacuum filtration system and glass rods.

Washing Powder/ Liquid Detergent, Disinfectants

Powder or liquid detergents or wetting agents such as Tween-20, 70% alcohol and aboslute alcohol ). Glass distilled water, Stock solutions of nutrients of tissue culture media or ready-made/prepared powdered media, Sucrose, Agar (tissue culture grade), Sterile culture vessels with distilled water. Chemicals of analytical grades (Inorganic, organic salts, vitamins, amino acids, growth tors/hormones and activated charcoal), Coconut milk, yeast extract, malt extract, casein lysate and extracts of potato, carrot and tomato.

Spatulas, weighing butter paper/boats, stirring bars (magnetic) for magnetic stirrers and stirring rods, Brush (flask and test tubes), gloves (disposable 23 cm), mop (household), scoop, towels Centrifuge (low speed): Variable electronic speed contro speed indicator, Amp. meter, timer, dynamic break, starting switch, 230V 50Hz.

Electric Autoclaves -Vertical: with safety valves, pressure gauge, steam-release cock.

Horizontal: mounted to tubular stand. heavy hings

Steamers: With immersion heaters of ejection safety device, size: 31 cm x 13cm x l0cm

to 61cm x 20cm x 15cm.

Filter Sterilization Equipment: Syringe filter holder: 2.5cm -Pressure Filter Holder: 4.7cm

Double Distilled Water Equipment: Built-in energy regulator all glass water stills, heat re-

sistant boiling flask with heater .

Environmental Growth Cabinets: Cabinet with controlled temperature, light and humidity, temperature range 4° to 45°C with timer to regulate photoperiods.

Gyratory Shakers: Capacity: 50 to 1000 ml flasks, speed: 80 to 200 rpm.

Laminar Air-flow Cabinets: Constant flow of purified air, sizes: 0.6m, 1.2 m and 1.8m.

The other miscellaneous equipments which are required for tissue culture are: air conditioners, arrow heads, bunsen bummers, deep-freeze, dissecting needles, glasswares, forceps, florescent lamps/tubes, heaters, hot plates with magnetic stirrer, inoculation cabinets, metal trays and bowls (for transport of cultures), tubes, refrigerators, UV germicidal lamps, wooden or metal racks, etc.

Aseptic Technique

In in vitro condition plant cells and microbes have basically same requirements. When the culture medium contains sugar (as carbon source) it attracts a variety of microorganisms which grow fast than that of the cultured tissue in medium and they ultimately kill the plant cells. It is, therefore, necessary to have complete aseptic condition around the culture equipments which prevents contamination of the culture medium. Following are the three main, sources of contamination of the medium and the subsequent methods to check them:

1. The microorganisms may be present in the nutrient medium at the time of its preparation. These microorganisms can be destroyed by proper plugging and autoclaving the culture tubes/flask. The medium can be completely sterilized by maintaining it at 120° C for about 20 minutes at 15 Ib pressure in the autoclave.

2. The explant (plant part to be cultured) may carry microorganisms with it, therefore, the excised part should be surface-sterilized by mercuric chloride (1 to 2%) or by sodium hypochlorite solution for 30 minutes.

Precautions must be observed to prevent the entry of microorganisms when the plug of theculture is removed during transfer of the plant material to the medium or from one medium to another .The inoculation chamber may be sterilized by UV -radiations.

Correct pH of the medium is important. Highly alkaline or acidic pH affects the nutrient up- take in culture tissues. Therefore, the tissue culture medium is adjusted to a pH of 5.6 to 6.0, before autoclaving.

Semi-solid and liquid media are most commonly used for growing plant cells. A high concentration of gelling agent (agar-agar, gelatin, silica gel) makes the medium very hard and de- creases the nutrient uptake by the tissues. Agar at 0.8% to 1,0% concentration is widely used.

It is essential to remove dirt and debris from the plant tissue and should be washed in a weak detergent solution and rinsed several times with distilled water prior to sterilization, Some woody trees, such as buds arid twigs, are cleaned by immersing them briefly in a 70% ethanol solution, 'mulch wets and spreads over the tissue surfaces more effectively than a higher concentration alcohol.

Sodium Hypochlorite (NaOCI): It is the most common chemical agent used to sterilize plant tissues (0.025%-0.25% NaOCI). Diluted house hold bleach can also be used for this purpose, which normally contains 5,25% NaOCl. It is equally effective and considerably less expensive, Calcium hypochlorite (CaOCI): It can be used as a substitute for NaOCI. CaOCI causes slightly less damage to plant tissues but tends to precipitate out of solution. To avoid the accumulation of CaOCI on the plant tissue surfaces, sterilization solutions should be filtered or decanted prior to use.

Hydrogen Peroxide (H20V Solution: Plant tissues can also be surface sterilized using a H202 (3%-10%). It is much easier to remove from tissues than NaOCI and CaOCI:

Other Substances: Plant tissue can also be surface-sterilized by bromine water (1 %-2%), silver nitrate (AgNO3 I %) and mercuric chloride (MgCI2 0.1 %-I %),

Cleaning (Preparation of Glasswares/Plastic wares (Autoclavable}

Clean the glasswares/plastic wares in 10% commercial detergent liquid or powder for I hr and then in HCI for 2 hr. Remove the traces of detergent and acid by thorough washing with tap water. Rinse vessels with double distilled water and allow them to dry over night at room temperature.

Plug glasswares such as conical flasks or test tubes with non-absorbent cotton or cover by the plastic caps, Wrap the petri dishes with aluminium foil. Place forceps and scalpels in test tubes, plug the tubes with cotton or cover with aluminium foil. Plug the mouth end of the pipettes with cotton. Wrap them individually in aluminium foil.