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Plant callus (plural calluses or calli) is a mass of unorganized parenchyma cells derived from plant tissue (explants) for use in biological research and biotechnology. In plant biology, callus cells are those cells that cover a plant wound. Callus formation is induced from plant tissues after surface sterilization and plating onto in vitro tissue culture medium. Plant growth regulators, such as auxins, cytokinins, and gibberellins, are supplemented into the medium to initiate callus formation or somatic embryogenesis. Callus tissue initiation has been described for many plant taxonomic divisions: Callus induction and tissue culture: A callus cell culture is usually sustained on gel medium. Callus induction medium consists of agar and a mixture of macronutrients and micronutrients for the given cell type. There are several types of basal salt mixtures used in plant tissue culture, but most notably modified Murashige and Skoog medium, White's medium, and woody plant medium. Vitamins are also provided to enhance growth such as Gamborg B5 vitamins. For plant cells, enrichment with nitrogen, phosphorus, and potassium is especially important.
Figure 1:callus formation
Morphology: Plant callus is usually derived from somatic tissues. The tissues used to initiate callus formation depends on plant species and which tissues are available for explant culture. The cells that give rise to callus and somatic embryos usually undergo rapid division or are
Ammocallis rosea. roseus Binomial name :Catharanthus roseus Synonyms: Vinca rosea Catharanthus roseus (Madagascar Periwinkle) is a species of Catharanthus native and endemic to Madagascar. but found throughout India. it is an endangered plant. Distribution: The plant is a native of Madagascar. In the wild. Synonyms include Vince rosea (the basionym). and Lochnera rosea. other English names occasionally used include Cape Periwinkle. Rose Periwinkle. Rosy Periwinkle. leaves are shinning green in clusters. flowers are white or pink in colour. the main cause of decline is habitat destruction by slash and burn 18 . and "Old-maid".CATHARANTHUS ROSEUS: Figure 2 :Catheranthus roseus Scientific classification Kingdom: Plantae : Angiosperms : Eudicots : Asterids Order: Gentianales Family: Apocynaceae Genus: Catharanthus Species: C. Nature of classification of Catharanthus roseus: it is a herb up to one-meter height.
in the rotating picture.forensics. genetics and molecular biology to separate proteins according to their electrophoretic mobility (a function of the length of a polypeptide chain and its charge) and no other physical feature. where the centripetal acceleration causes denser substances to separate out along the radial direction (the bottom of the tube). Centrifuge: A centrifuge is a piece of equipment. sodium dodecyl sulfate polyacrylamide gel electrophoresis. Figure 3:Centrifuge In the picture shown. called the rotor. that puts an object in rotation around a fixed axis. As the centrifugal force is in the horizontal plane and the tubes are fixed at an angle. II. SDS. the rotating unit. the particles have to travel only a little distance before they hit the wall and drop down to the bottom. These angle rotors are very popular in the lab for routine use. generally driven by an electric motor (some older models were spun by hand). describes a technique widely used in biochemistry. applying a force perpendicular to the axis. SDS is an anionic 40 . Test tubes are placed in these slots and the motor is spun.INSTRUMENTS USED: I. The centrifuge works using the sedimentation principle.PAGE: SDS-PAGE. has fixed holes drilled at an angle (to the vertical). move to the centre). By the same token lighter objects will tend to move to the top (of the tube.
what substances are present in a target and exactly how much through calculations of observed wavelengths.detergent applied to protein sample to linearize proteins and to impart a negative charge to linearized proteins. calibrations are needed on the machine using standards that vary in type depending on the wavelength of the photometric determination. However they can also be designed to measure the diffusivity on any of the listed light ranges that usually cover around 200nm . IV. Water bath Eppendorf tube Spectrophotometer: A spectrophotometer is a photometer that can measure intensity as a function of the light source wavelength. depending on the control or calibration. Within these ranges of light. Spectrophotometer is able to determine. A spectrophotometer is commonly used for the measurement of transmittance or reflectance of solutions. In most proteins. such as polished glass.2500nm using different controls and calibrations. the binding of SDS to the polypeptide chain imparts an even distribution of charge per unit mass. Figure 4: SDS-PAGE III. thereby resulting in a fractionation by approximate size during electrophoresis. Important features of spectrophotometers are spectral bandwidth and linear range of absorption or reflectance measurement. 41 . V. transparent or opaque solids. or gases.
Figure 5:spectrophotometer VI. Beakers/Glasswares Micropipette tips Micropipette Transilluminator Oven pH meter: A pH meter is an electronic instrument used for measuring the pH (acidity or alkalinity) of a liquid (though special probes are sometimes used to measure the pH of semi-solid substances). XI. Laminar air flow Weighing machine 42 . IX. VIII. XIII. A typical pH meter consists of a special measuring probe (a glass electrode) connected to an electronic meter that measures and displays the pH reading Figure 6: ph meter XII. X. VII.
roseus D: Maximum multiple shoot formation from hypocotyl derived calli in C.A: Initiation of Callus formation in C. roseus F: Root formed were hard. roseus G: Transfer of regenerated plantlets into pot containing Farmyard Manure H: Survived plantlets with morphologically normal healthy stem and leaves in C.5 mg/l) in C. thick and hairy in C. roseus C: Initiation of multiple shoot formation on shooting medium in C. roseus E: Maximum root induction on rooting medium supplemented with IBA (2.5mg/l) + NAA (0. Roseus 57 . roseus B: Shoot regeneration from hypocotyl derived calli in C.
672 0. 2. 5. Callus is crushed and then centrifuged at 10.798 0.NO.378 0. Under in-vitro condition we use callus as a sample. OD is measured using spectrophotometer at 560nm.804 STEM 0. 3. Activity of invertase in in-vitro catheranthus roseus is maximum on 45th day and least on 15th day.613 0. Activity of invertase in in-vivo catheranthus roseus is maximum in leaf and least in root. 4.628 0.802 0.402 0.439 0. 1.000rpm.406 Table 3:Invertase(in-vivo) 1. 58 .813 0. 2.637 ROOT 0. 3.Comparision of invertase enzyme activity: INVERTASE (IN-VIVO) S. TOTAL LEAF 0.
TOTAL 15 DAY 0.498 0.578 0.512 0.480 45 DAY 0.562 Table 4:Invertase (in-vitro) 59 .NO.465 0.326 0.478 0. 1.INVERTASE (IN-VITRO).598 0.CALLUS TAKEN S.395 0.373 30 DAY 0.389 0. 2. 3.
912 1. 1.710 0.872 0. Callus is crushed and then centrifuged at 10.762 0.474 Table 5:catalase(in-vivo) 1. 3. 2. Activity of catalase in in-vivo cateranthus roseus is maximum in root and least in stem.Comparison of catalase enzyme activity: CATALASE (IN-VIVO) S. OD is measured using spectrophotometer at 560nm.524 0.987 1.000rpm.NO. Activity of catalase in in-vitro cateranthus roseus is maximum on 45th day and least on 15th day.723 ROOT 1. Under in-vitro condition we use callus as a sample. 4.974 0.925 STEM 0.931 0. 60 .698 0. TOTAL LEAF 0. 3. 5. 2.
715 0.712 45 DAY 0.892 0.912 0.628 0.720 0.608 30 DAY 0. 1.606 0.701 0.875 Table 6:catalase(in-vitro) 61 . TOTAL 15 DAY 0.823 0.CALLUS TAKEN S.CATALASE (IN-VITRO). 3. 2.592 0.NO.
2. leaves and roots 3.Comparison of peroxidise activity: Figure 7 : Peroxidase activity 1. 62 . Different bands are obtained showing amount of peroxidase in stems. Enzymatic activity of peroxidase is determined by using SDS-PAGE. Peroxidase activity is more in roots in comparison to stems and leaves.
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