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SEMINAR ON CRYOPRESERVATION SUBMITTED BY: Vinit parmar M.pharm-I Roll No. 090605014 Dept. of Pharmacognosy M.C.O.P.S, Manipal.

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Brit mom gives birth to '80s boy! TIMES NEWS NETWORK [ TUESDAY, MAY 25, 2004 05:02:07 PM ] The healthy baby boy, from 21-yearold sperm, is thought to be the world’s first instance of long-term freezing ending in a live birth. Dr Elizabeth Pease, a consultant in reproductive medicine at the Manchester hospital, said the development is "important because we believe this is the longest period of sperm cryopreservation resulting in a live birth so far reported in the scientific literature".

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What is cryopreservation? Cryobiology is the study of the effects of extremely low temperatures on biological systems, such as cells or organisms. Cryopreservation—“Cryopreservation is defined as the viable freezing of biological material and their subsequent storage at ultra low temperatures (-1960C)”.

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In the recent years, with tremendous increase in population, pressure on the forest and land resources have increased, which in turn caused decrease in the population of medicinal and aromatic plant species. Even some are on verge of extinction. Not only the plants but also some animal species are also on verge of extinction. Thus cryopreservation can be used as an effective means to conserve the germplasms of such species.

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CONVENTIONAL METHODS VS. CRYOPRESERVATION -Conventional methods of storage or preservation of germplasm has some limitations, they are expensive and time consuming. -A cell suspension culture is needed to be subcultured every 7 to 10 days and callus cultures every 14 to 30 days. Furthermore, there is risk of possible loss through contamination or equipment failure.

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-Liquid nitrogen is most widely used material for cryopreservation. Dry ice can also be used. Why liquid nitrogen? • Chemically inert • Relatively low cost • Non-toxic • Non-flammable • Readily available

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STEPS INVOLVED IN CRYOPRESERVATION: 1. Selection of plant material 2. Pregrowth 3. Addition of cryoprotectants Vitrification Cryoprotective dehydration Encapsulation and dehydration 4. Freezing Rapid freezing Slow freezing Stepwise freezing

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5. Storage 6. Thawing 7. Determation of survival/ viability -TTC method of staining. -Evans blue staining. -Fluorescien diacetate [FDA] staining.



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-Morphological and physiological conditions of plant material influence the ability of explants to survive during cryopreservation. Factors which may be considered are; -Tissue must be selected from the healthy plants. -In general small, young, rich in cytoplasm, meristematic cells survive better than the larger, highly vacuolated cells. -Callus derived from tropical plant is more resistant to freezing damage. -Water content of cell or tissue used for cryopreservation should be low because with low freezable water, tissues can withstand extremely low temperature.

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2. Pregrowth: -Pregrowth treatment protect plant tissues against exposure to liquid nitrogen. -Pregrowth involves the application of additives known to enhance plant stress tolerance. E.g. abscisic acid, praline, trehalose. -Partial tissue dehydration can be achieved by the application of osmotically active compounds. E.g. i] C. roseus cells are precultured in medium containing 1M sorbitol before freezing. ii] Digitalis cells were precultured on 6% mannitol medium for 3 days before freezing.

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3. Addition of cryoprotectants: -There are two potential sources of cell damage during cryopreservation. i] Formation of large ice crystals, inside the cell. ii] Intracellular concentration of solutes increase to toxic levels before or during freezing as a result of dehydration. Cryoprotectants acts like antifreeze; they lower freezing temperature and increase viscosity.

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HOW CRYOPROTECTANTS HELP IN PREVENTING DAMAGE TO THE CELLS; a] Vitrification: It is process in which ice formation can not take place because the aqueous solution is too concentrated to permit ice crystals nucleation. Instead, water solidifies into an amorphous ‘glassy’ state.

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b] Cryoprotective dehydration: If cells are sufficiently dehydrated they may be able to withstand immersion in liquid nitrogen. Dehydration can be achieved by growth in presence of high concentration of osmotically active compounds like sugars, polyols and / or air desiccation in a sterile flow cabinet or over silica gel. Dehydration reduces the amount of water available for the ice formation. Dehydration increases the osmotic pressure of the intracellular solution (the cytoplasm) which depresses its freezing temperature and promotes vitrification---both inhibit intracellular ice formation.

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c] Encapsulation and dehydration: -This involves the encapsulation of tissues in calcium alginate beads which are pregrown in liquid culture media containing high concentration of sucrose. -After these treatments the tissues are able to withstand exposure to liquid nitrogen without application of chemical cryoprotectants.

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Various cryoprotectants used are glycerol, dimethyl sulphoxide, mannitol, propylene glycol etc. DMSO is an excellent cryoprotectant, it is non-toxic, easily permeable and having low molecular weight and is easily washable from the cells. -It is used in concentration of 5 to 10 %.

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4. FREEZING: -Freezing can be achieved in three different ways; 1.Rapid freezing: -The material is placed in vials and plunged into liquid nitrogen and decrease in temperature from -300 to -10000C/ MIN. OR MORE OCCURS. -The quicker the freezing is done, the smaller the intracellular ice crystals are. -Rapid freezing has been employed for cryopreservation of shoot tips of potato, strawberry, brassica species.

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2. Slow freezing: -Tissue is slowly frozen with decrease in temperature from -0.1 to 100C/min. -Slow cooling permits the flow of water from the cells to the outside, thereby promoting extracellular ice formation instead of lethal intracellular freezing. - This method has been successfully employed for cryopreservation of meristems of peas, potato, cassava, strawberry etc.

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3. Stepwise freezing: -In this method slow freezing down to -20 to -400C, a stop for a period of approximately 30 min. and then additional rapid freezing to – 1960C is done by plunging in liquid nitrogen. 5. STORAGE: Storage of frozen material at the correct temperature is as important as freezing. -The frozen cells / tissues are kept for storage at temperature ranging from – 70 to -1960 C. -Temperature should be sufficiently low for long term storage of cells to stop all metabolic activities and prevent biochemical injury. -Long term storage is best done at – 1960C.

6. THAWING:- Bringing back…. : 

6. THAWING:- Bringing back….

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It is done by putting the ampoule containing the sample in a warm water bath [350 to 400c]. -Frozen tips of the sample in tubes or ampoules are plunged into the warm water with a vigorous swirling action just to the point of ice disappearance. -It is important for the survival of the tissue that the tubes should not be left in the warm water bath after ice melts. Just at point of thawing quickly transfer the tubes to a water bath maintained at room temperature and continue the swirling action for 15 sec. to cool, the warm walls of the tube. -Tissue which has been frozen by encapsulation/ dehydration is frequently thawed at ambient temperature.

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7. DETERMINATION OF SURVIVAL / VIABILITY: -Regrowth of the plants from stored tissues or cells is the only test of survival of plant materials. -Various viability tests include Fluorescien diacetate [FDA] staining, growth measurement by cell number, dry and fresh weight. -Important staining methods are triphenyl tetrazolium chloride [TTC], Evan’s blue staining.

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A. TTC assay: Cell survival is measured by amount of red formazan product formed due to reduction of triphenyl tetrazolium chloride [TTC] which is measured spectrometrically. Only the viable cells which contain the enzyme mitochondrial dehydrogenase which reduces TTC to red formazan will be stained and dead cells will not take up the dye.

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B. Evan’s blue staining: -One drop of 0.1% solution of Evan’s blue is added to cell suspension on a microscope slide and observed under light microscope. only Non viable cells [dead cells] stain with Evan’s blue. % of viable cells= No. of fluorescent cells Х 100 total no. of cells (Viable + non-viable)

Individual cell viability assayed with Evan’s blue dye and fluorescein diacetate. : 

Individual cell viability assayed with Evan’s blue dye and fluorescein diacetate.

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APPLICATIONS OF CRYOPRESERVATION: 1] Conservation of genetic material: Cryopreservation provides an Opportunity for conservation of endangered medicinal plants. -For example, low temperature storage has been reported to be effective for cell cultures of medicinal and alkaloid producing plants such as Rauwolfia serpentina, D. lanata, A. belladonna, Hyoscyamus spp. -Cryopreservation has been used successfully to store a range of tissue types, including meristems, anthers/pollens and embryos.

Plzz Cryopreserve our bodies…….. : 

Plzz Cryopreserve our bodies……..

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The living rate: • Various companies offer to freeze your body after death for potential reviving later. • Current prices: – Whole body- $1500/yr – Head only- $250/yr – Brain only- $200/yr – "Perpetual storage"- $120,000

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2] Freeze storage of cell cultures: A cell line to be maintained has to be subcultured and transferred periodically and repeatedly over an extended period of time. Cryopreservation is an ideal approach to suppress cell division to avoid the need for periodical subculturing. 3] Maintenance of disease free stock: Pathogen free stocks of rare plant material could be frozen and propagated when needed. 4] Cold acclimatization and frost resistance: A cryopreserved tissue culture would provide a suitable material for selection of cold resistant mutant cell lines, which could later differentiate into frost resistance plants.

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5] Application of cryopreservation to herbaceous species: A] Cell suspension and callus culture: Cell suspension and callus culture are often cryopreserved using classical slow cooling protocols for the conservation of specific features of tissue that can be lost during normal invitro maintenance. B] Cryopreservation of seeds: Although the most economical means of germplasm storage for seed propagated species is in the form of seeds, this is not always feasible because of the following reasons: -Some crops do not produce viable seeds -Some seeds remain viable for a limited duration only and are recalcitrant to storage. -Seeds of certain species deteriorate rapidly due to seed borne pathogen -Some seeds are very heterozygous not suitable for maintaining true to type genotypes. -Effective approach to circumvent the above problems may be application of cryopreservation technology. Celery is an example of orthodox seed cryopreservation.

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REFERENCES: 1. R.A.Dixon, R.A.Gonzales, Plant cell culture, a practical approach, IInd edition, page no. 147-151. 2. Ciddi veeresham, Medicinal plant biotechnology, CBS publishers &distributors, 1st edition, 2006, page no. 46, 486-492. 3. John H.Dodds & Lorin W. Roberts, Experiments in plant tissue culture, IIIrd edition, page no. 195-198. 4. Dr. P.E.Rajashekhran, Presentation on cryopreservation of plant systems: challenges and prospects, Indian Institute of Horticultural Research, Hessaraghatta lake, BANGALORE.

THANK U …. Hope your minds are not FROZEN… If they are then please remember THAWING…. : 

THANK U …. Hope your minds are not FROZEN… If they are then please remember THAWING….

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