Micropropagation tools

The culture or a portion of the culture is removed from the jar and placed on a sterile paper towel. A scalpel and forceps are used to cut and separate the larger culture into smaller pieces for transfer to a new jar to complete the subculturing procedure. Click on the button below to see Stage II movie.

A high cytokinin to auxin ratio is used during the multiplication stage to induce axillary or adventitious shoot formation. Too high a concentration of cytokinin will result in a high number of adventitious shoots that do not elongate.

Shoots multiplied in culture must be rooted in Stage III in order to create a new plantlet. In the rooting stage, microcuttings are induced to form roots - usually by application of auxin. In general, species root easier in tissue culture than they do from conventional cuttings.

In general, microcuttings rooted ex vitro have a more normal root system and acclimatize to a normal growing environment better than cuttings rooted in vitro. However, the propagator has more control over the rooting environment in vitro and this method may fit their production scheme better. Microcuttings are inserted directly into the rooting substrate often using forceps to handle the small cuttings. At each work station the technicians have their rooting flats, a syringe bottle to spray microcuttings periodically to keep them from drying out.

Technicians sticking microcuttings at a workstation. Microcuttings after the agar has been washed off. The cell culture and callus culture are done for the production of secondary metabolites such as flavours, fragrance, essential oils, pigments, alkaloids, etc. It involves the fusion of protoplasts of two plant cells of different species.

This fusion of protoplasts of unrelated species results in the development of hybrids called somatic hybrids. Example: Pomato is a somatic hybrid of potato and tomato. Some of the limitations or disadvantages of micropropagation are as follows: i.

Micropropagation techniques require intensive labour, and this often limits their commercial application. Automation can reduce the labour required. The plants grown from this method find a problem in acclimatizing to the new natural environment. During the micropropagation, several slow-growing microorganisms contaminate and grow in cultures. Attempt Mock Tests.

Micropropagation is an advanced artificial vegetative propagation technique. This method helps in getting disease-free plants in very little time. This method also does not require more space and can produce a large number of plantlets using only tissue, organs or any part of the plant. What do you mean by micropropagation?

Ans: In tissue culture, millions of agricultural and horticulture plants are multiplied. This is called micropropagation. Write two advantages of micropropagation?

Ans: The two advantages of micropropagation are as follows: i. Large numbers of plants can be grown in a very short time and less space. A disease-free plant variety can be produced using micropropagation. What are the steps of micropropagation? Ans: The steps or stages of micropropagation are: i. Selection of an explant ii. Culture initiation and establishment iii.

Shoot multiplication iv. Rooting of the shoots v. Transfer of plantlets in the greenhouse environment. What is macropropagation? This is used to determine concentration in fractions obtained in column chromatography, TLC and in other solutions. Identification and structure evaluation of various isolated pure compounds can be done by UV-Visible spectrophotometry. All the compounds have very specific absorption spectra depending on the molecular structure.

Comparison of spectra helps in identification of compounds. Spectrophotometers coupled with microprocessor or computer can store data, figures and are helpful in comparing the effect of different variables. A centrifuge is an instrument which produces centrifugal force by rotating the samples around a central axis with the help of an electric motor. Centrifuges can be categorized as the clinical type , rpm , refrigerated high-speed centrifuges 10,, rpm and ultra -centrifuges 20, to 80, rpm.

With increase in rpm, the friction of rotor with air produces so much of heat that they have to be run under refrigeration so called refrigerated centrifuge and both refrigeration and vacuum are used in ultracentrifuge, which runs at very high rpm.

For these high speeds, even the rotor has to be made of special metal to withstand the great force. There are two types of rotors, angle head and swing Fig. Simple calculations show that for the same radius, the swinging bucket method produces more gravitational force. Ultracentrifuges are of two types — analytical and preparative model.

This consists of rotors and tubes, called cells. The instrument is designed to allow the operator to follow the progress of the substances in the cells, while the process of centrifugation is in progress. By estimating sedimentation velocity during the process, the molecular weight, purity etc. This is used for purification of the components of macromolecules or other substances and all determinations are made at the end of centrifugation.

The instrument has no monitoring device, while large centrifugal forces are set for a fixed time period. Centrifugation is the most widely used technique for separation of various metabolites and also used to separate non-miscible liquids during extraction of secondary metabolites, e. A wide variety of centrifuges are available, ranging in capacity and speed. During the process of centrifugation, solid particles experience a centrifugal force, which pulls them outwards, i.

The velocity with which a given solid particle moves through a liquid medium is related to angular velocity. The sedimentation coefficient is a characteristic constant for a molecule or a particle and is a function of the size, shape and density. It is equivalent to the average velocity per unit of acceleration. The unit Svedberg s is often used with reference to centrifugation and is equivalent to a sedimentation coefficient of 10 s.

Centrifugation is widely used in analytical techniques, preparation of extracts, separation of non-miscible liquid mixtures, purification of enzymes, inhibitors and removing particles silica, etc.

Centrifugation with heating and vacuum suction is used for concentrating extraction instrument called sample concentrator and rapidly removing solvents.

All the enzymatic work requires refrigerated centrifuges to keep the samples cool during centrifugation and to protect enzymes from inactivation by heat. The term chromatography was used by the Russian botanist Tswett to describe the separation of plant pigments on a column of alumina. There are different types of chromatography but they all involve interactions between these components: the mixture to be separated, a solid phase, and a solvent. The magnitude of these interactions depends upon the particular method used.

Usually column chromatography is used to separate large quantities of compounds, whereas, paper chromatography PC or thin-layer chromatography TLC in one or two dimensions, is used for analytical work. The mobile phase can be a gas or a liquid, whereas the stationary phase can only be a liquid or solid.

In liquid column chromatography LCC , separation involves a simple partitioning between two immiscible liquid phases, one stationary and the other mobile, the process is called liquid-liquid or partition chromatography LLC. When physical surface forces are mainly involved in the retentive ability of the stationary phase, the process is denoted liquid-solid or adsorption chromatography LSC. In ion exchange chromatography IEC , ionic components of the sample are separated by selective exchange with counter ions of the stationary phase.

Cellulose in the form of paper sheets makes an ideal support medium where water is absorbed between the cellulose fibres and forms a stationary hydrophilic phase. The suitably concentrated mixture is spotted onto the paper, dried with a hair-drier and the chromatogram is developed by allowing the solvent to flow along the sheet.

The solvent front is marked and after drying the paper, the positions of the compounds present in the mixture are visualized by a suitable staining reaction. The ratio of the distance moved by a compound to that moved by the solvent is known as the R ; value and is more or less constant for a particular compound, solvent system and paper under carefully controlled conditions of solute concentration, temperature and pH.

Generally, alcoholic extracts of plant material with or without partial purification are used for chromatography. Biological materials should be desalted before chromatography by electrolysis or electro-dialysis. Excess salt results in a poor chromatogram with spreading of spots and changes in their R.

It can also affect the chemical reactions used to detect the compounds being separated. Whatman No. For rapid separation, Whatman Nos. The paper may be impregnated with a buffer solution before use or chemically modified by acetylation. Ion exchange papers are also available commercially. For the separation of lipids and similar hydrophobic molecules, silica-impregnated papers are available commercially.

This choice, like that of the paper, is largely empirical and will depend on the mixture investigated. This value is a constant for a particular compound under standard conditions and closely reflects the distribution coefficient for that compound. It is recommended that the developing chamber should be saturated with solvent, by using filter paper lining inside the chamber.

After location, a map is obtained and compounds can be identified by comparing their position with a map of known compounds developed under the same conditions Fig. Most compounds are colourless and are visualized by specific reagents. The location reagent is applied by spraying the paper under a fumigation hood or rapidly dipping it in a solution of the reagent in a volatile solvent. Viewing under ultraviolet light is also useful since some compounds, which absorb strongly show up as dark spots against the fluorescent background of the paper.

Other compounds show a characteristic fluorescence under ultraviolet light. Separation of compounds on a thin layer of adsorbing material is similar in many ways to paper chromatography, but has the added advantage that a variety of supporting media can be used so that separation can be by adsorption ion exchange, partition chromatography, or gel filtration depending on the nature of the medium employed.

The method is very rapid compared to paper chromatography and many separations can be completed within an hour. Compounds can be detected at a lower concentration than on paper as the spots are very compact. Furthermore, separated compounds can be detected by corrosive sprays and elevated temperatures with some thin layer materials, which of course is not possible with paper. There are several good spreaders available in the market, which can produce an even layer of required thickness by adjusting the thickness control screw.

Calcium sulphate is sometimes incorporated into the adsorbent to bind the layer to the plate and, because of this; it is advisable to work rapidly once the adsorbent is mixed with water. There are now a number of prepared thin layer plates using different adsorbents on various supporting materials such as glass, plastic, and aluminum that are available commercially and these may be more convenient to use than trying to prepare plates in the laboratory.

However, horizontal chambers of high performance TLC HPTLC are very useful and convenient as they require less time, solvent and no stabilization time. Development of the plate is usually by the ascending technique and is very rapid. The thermometer is a device that measures temperature or temperature gradient using a variety of different principles; it comes from the Greek roots thermo, heat, and meter, to measure.

A thermometer has two important elements: the temperature sensor e. Industrial thermometers commonly use electronic means to provide a digital display or input to a computer. The Alcohol thermometer or Spirit thermometer is an alternative to the Mercury-in-glass thermometer, and functions in a similar way. An organic liquid is contained in a glass bulb which is connected to a capillary of the same glass and the end is sealed with an expansion bulb.

The space above the liquid is a mixture of nitrogen and the vapour of the liquid. For the working temperature range, the meniscus or interface between the liquid is within the capillary. With increasing temperature, the volume of liquid expands and the meniscus moves up the capillary. The position of the meniscus shows the temperature against an inscribed scale.

The liquid used can be pure ethanol or toluene or kerosene or Isoamyl acetate, depending on manufacturer and working temperature range. Since these are transparent, the liquid is made more visible by the addition of a red or blue dye. One half of the glass containing the capillary is usually enameled white or yellow to give a background for reading the scale.

Temperature is measured by maximum-minimum thermometer or a continuous rotary dram chart type thermometer. The U- shaped maximum-minimum thermometer is commonly used for determining diurnal maximum and minimum range of temperature in the culture room. Therefore, chokes of the tube lights are fitted outside the culture room.

The indicators of the maximum-minimum thermometer are moved by mercury column and they remain at that position until moved by the observer with the help of magnet. Their positions indicate the minimum and maximum temperature in the previous 24 h. After recording temperature indicators are reset to mercury level Fig. Hygrometers are instruments used for measuring humidity. Evaporation from the wet bulb lowers the temperature, so that the wet-bulb thermometer usually shows a lower temperature than that of the dry-bulb thermometer, which measures dry-bulb temperature.

Relative humidity is computed from the ambient temperature as shown by the dry- bulb thermometer and the difference in temperatures as shown by the wet-bulb and dry-bulb thermometers. Relative humidity can also be determined by locating the intersection of the wet- and dry-bulb temperatures on a psychrometric chart.

Dial type hair hygrometer is a convenient tool to measure humidity in the culture room. Humidistat is a bimetallic thermocouple device to control and regulate the function of humidifier to maintain the humidity. Distilled water should be filled in the humidifier. The RH present in the culture room is measured by hair hygrometer. As the name suggests a chemically treated hair elongates with increased humidity and shortens with dryness similar to mercury in thermometer.

At lower humidity, medium dries rapidly whereas at higher humidity chances of fungal growth over all surfaces and cotton plugs is increased.

Light is the form of radiant energy, i. Visible light as we perceive, is located in narrow wavelength region of spectrum between to nm. Light has dual characters, displaying both wave properties refraction, diffraction, interference and polarization phenomena and particle properties light is radiated in discrete amounts of energy or photons.

It is to note that an irradiance measurement is not spectrally defined, whereas, an illuminance measurement indicates the level of visible light as the human eye would see it. Light intensity can be measured by photometer or lux meter.

A photometer consists of photoelectric cell and a micro-ammeter. Photoelectric cell is sensitive for light and converts light into current. Micro-ammeter shows reading due to this current and its needle moves. Nowadays digital read out is given by appropriately converting the current into digital signal Fig. High intensity is proportional to the current generated in the photoelectric cell by falling light.

Usually to lux is provided to the cultures maintained in light in the culture room. The instrument is a sensitive tool and handled with care. It should not be exposed to the sunlight without switching the proper reading switch to high light illumination.

Plant tissue culture requires contamination free environment, tools and cultures or strict maintenance of germ free system in all the operations, known as asepsis.

Particularly in commercial production units, the contamination of one batch of the cultures may result in heavy financial losses or even loss of a culture strain. Therefore strict control measures are enforced to maintain the entry of the personnel and living materials.

The basic rules and practices of asepsis are followed in all the tissue culture laboratories. Plant tissue culture media are rich in nutrients and very suitable for the growth of microbes also. These microorganisms grow faster, consume nutrients rapidly and suppress the growth of plant tissues by over growth. This saprophytic unwanted growth of microbes is formed as contamination. There are many ways by which these microbes suppress the growth of plant cells and tissues, e.

To achieve success in cultivation of higher plant parts, it is essential to exclude these contaminating microorganisms and hence aseptic techniques must be employed to save cultures. All the area, tools and working places should be free from microbes to have less and less problems of contamination. Minimize the air current in the working area so it is possible to avoid spores of contaminating microorganisms to move in along with the air currents over the sterile areas.

At least, fan should not be used in laminar air flow bench room inoculation room. Preferably, all the places should be air-conditioned. UV tubes are also fixed in Laminar air flow bench placed in inoculation chamber. All these UV tube lights should be used frequently before inoculations. UV lights of corridors may be left open during nights.

Most contamination is introduced with the explant because of inadequate sterilization or just very dirty material. It can be fungal or bacterial. This kind of contamination can be a very difficult problem when the plant explants material is harvested from the field or greenhouse. Initial contamination is obvious within a few days after cultures are initiated. Bacteria are the most frequent contaminants.

They are usually introduced with the explants and may survive surface sterilization of the explants because they are in interior tissues. So, bacterial contamination can first become apparent long after a culture has been initiated. All explants have to be sterilized before transferring them on to the medium. Sterilized petridishes, distilled water, scalpel, filter paper sheets suitable sized and autoclaved alcohol, disinfectant mercuric chloride 0.

Put on the UV light of the bench and put it off after 30 minutes.