Introduction to fe eC] Mae] Pee em eure Deed eeu a eer Sel) Nea elCELL CULTURE = Culturing of single cells in vitro in an artificial nutrient media is known as cell culture. = Cell cultures are carried out to investigate potentialities and properties of individual cells. = Isolation of single cells: 1. From intact organ 2. From callus pe Deo | 2 — are Preteen ies sedIsolation of single cells: This can be achieved from intact organs and from the Cor] (0 From intact organ: Mechanical and enzymatic methods are used to isolate the cells from intact organs. » The plant part preferably leaf tissue is subjected for grinding in a mortar with pestle along with addition of grinding medium consisting of sucrose 20 pmol. MgCl, 10 mol, Tris Hydrochloride buffer 20 pmol (pH 7-8). The resulting mixture is subjected for centrifugation and cells are taken up into a liquid media. Mi at-we) lalla ersr hee Ri Lda lam -lePA7 tnt dae TA macerozyme along with 1% potassium dextran sulphate, which fe TW em en Melle cielo MolmuN ele ACTA Raa(Ik-a oy Vale Le are aa cells. Preteen ies sedThe enzyme also causes the weakening of cell wall thus creating the problems associated with osmosis. Hence the cells are provided with osmotic protection with a substance known as osmoticum. Commonly used osmoticums are sorbitol 450-800 mmol/l, KCI 335 mmol/l, Magnesium sulphate 40 mmol/l. bia cedar] (oo = Callus preferably friable callus upon agitation in suitable liquid media results in the dissociation of the cells from callus. © The agitation will also helps in uniform distribution, breaking up of lumps and gaseous exchange between culture air and atmosphere.Culturing of cells: = The cells obtained in the isolation will be subjected for culturing by which the cells build up quickly. = This can be achieved with batch and continuous cultures. = Batch Cultures: SM luelee mice men et) ato Kee) Oe MCAT Te RCo ali Lemay oF leel healt] eee © The duration of the lag phase depends on the growth phase of the cells in stock culture at the time of culturing or sub culturing cer Mela dt Minera) ame Rel elt OVe = Aninitial cell density of 9-15 x 103 cells /ml is the best.e=s-1ca ae) (eel Me elec lg em Ne Reet ea lale[eea pattern of cell growth and metabolism and also by change in the composition of the medium. © Inthese cultures, exponential growth with constancy of cell doubling time may be achieved, but there is o period of CLAUoNATE1e Meco WAM MMA MNC Le Meola ic deli eol metabolites and enzymes are also constant. Peres eaea(Gofal loene elec to The continuous cultures are also known as mass cultures. In these cultures the cell are never allowed to enter in to the Sieh aa Uae These are of closed continuous and open continuous cultures. Closed continuous culture-they are characterised by the addition of fresh medium and outflow of equal amount of old medium. Cell biomass continues to increase as the growth proceeds. Open continuous cultures are characterised by addition of fresh medium while harvesting the equal amount of the old medium. Miata) CoM area ieM nN alegre Rome LMR Reon Teale LAre| sub maximal growth. Preteen ies sednO) Ta Mae) [eel Mol Renato Te: Leen aU gel Ce Ce eem a Zo depending upon by which the old media is replaced with fresh media. * Asinculture mediai.e in actively growing suspension cultures, the inorganic phosphate is rapidly utilised and consequently it soon becomesa limiting factor. The amount of inorganic phosphate is the basis for chemostatic methods. = Inthe turbidistatic methods the turbidity of the culture is taken into consideration. The turbidity is preselected on the basis of biomass density and can be maintained by intermittent flow of medium and washout of cells. Preteen ies sedAssessment of cell growth: = Cell counting : In the known volume of cell culture the cells are counted using haemocytometer. © To facilitate the cell counting, the cell aggregates are treated with 5-8% chromic acid or 0.1-0.25% pectinase solutions, which causes the breaking up of lumps. SNe Ree La al ae) Cee Ua re LL a ad (ag meet acre Preteen ies sed= Packed cell volume: » To determine the PCV a known volume of the uniformly dispersed culture is transferred to a 15 ml graduated centrifuge tube, then subjected for centrifugation at 200xg sfoe wala » The sediment formed is measured and expressed as ml of iT laacel man Kelme iael ge = Cell wet weight: ® To determine the cell wet weight a known volume of the uniformly dispersed culture is collected on pre weighed circular filter of nylon fabric supported in hartley funnel. See Maen Red Teal CoB LEARNT CBA ON MGA) medium, under vacuum and weighed and represented as grams of cells/ml of culture. Preteen ies sed= Cell dry weight: ® To determine the cell dry weight a known volume of the uniformly dispersed culture is collected on pre weighed circular filter of CNY Cola) oLaCee10) 9) Lela eR Malla) aU aL Then the cells are washed with water to remove the medium, under vacuum and dried at 60°C for 12 hours and weighed & and represented as grams of cells/ml of culture. Preteen ies sed* Non invasive method: ® Inthis method, the culture flask is fitted with ruler, and is tilted at an angle of 30° to 60° for 5 min and the height of the sediment is reordered. Change in the height of the sediment with the age of culture would represent the change in fresh weight of cells. Preteen ies sedAssessment of cell viability: = Phase contrast microscopy: = Using phase contrast microscopy, the cytoplastic streaming and the presence of healthy nucleus will be observed. Thus the viable cells are counted for the given volume of culture.* Reduction of tetrazolium salts: » When the cell cultures are incubated with 2,3,5 triphenyl tetrazolium chloride (TTC), the viable cells converts the TTC into ared colored substance known as 1,3,5 tripheny! Formazan, which is estimated spectrophotometrically. ‘TPF (Red Color Preteen ies sed= Fluorescein diacetate (FDA) method: cy Cell cultures are incubated with 0.5% FDA for 5 min. FDA being non polar and non fluorescing, enters the cells CTR Re Joh VAN e- a et Levan M Nao cae Lea) into polar Fluorescein. Since Fluorescein is not freely permeable across the plasma membrane, it accumulates mainly in the cytoplasm of intact cells, thus those cells exhibit green colour fluorescence. Preteen ies sed Fe= Evans Blue method: cy Cell cultures are incubated with 0.025% Evans blue for 5 min. Evans blue dye has been used as a viability assay on the basis of its penetration into non-viable cells i.e dead and damaged {oc The dead or damaged cell takes the blue colour. Preteen ies sedCulturing of single cells = Bergmann’s cell plating technique: » The most popular technique of single cell culture is Bergmann's cell plating technique. © Suspension cultures which carry cell aggregates in addition to free cells should be filtered through a sieve which would allow only single cells and small cell aggregates to pass through. The large cell aggregates are discarded and only the fine suspension is plated. © Free cells are suspended in a liquid medium at a density twice the finally desired plating cell density. » Melted agar (0.6-1%)-containing medium of otherwise the same composition as the liquid medium is cooled and maintained at 35°C in a water bath. Preteen ies sedEqual volumes of the two media are mixed and rapidly spread out in Petri dishes in such a manner that the cells ETc RMI VAC Lele) ccteR- nom ih for 10 days (KeeBeesiileYelmal Mae ills) Microbe free plants can be best produced through shoot tip Cav eee One important method of vegetative propagation. About 10mm long shoots are removed from healthy plants and these explants are subjected to surface sterilization in hypochlorite solution for 15 min. Then the distilled water washed explants are placed on petri-dish lined with moistened filter paper for dissecting the shoot apex. The terminal 0.5mm of the shoot apex is carefully excised and is placed culture vessel containing the cultured medium. Then the vessels are covered non absorbent cotton followed by aluminium foil and subjected for incubation at 27°C with light intensity of 1000 lux at 70% relative humidity for 16 oles Preteen ies sedAfter formation of the callus, the callus transformed on to the medium with high cytokinin concentration to induce shoots followed by high auxin concentration to induce roots om leelan ig. 8. Caltare of hoot ip of reid Combo Shoot tp placed on culate medium, ‘bCallsietormed, Alternatively the tips of the shoots can be cultured on the medium, resulting in the production of clumps of shoots from either axillary or adventitious buds. This method is known as micro propagation. Preteen ies sedPreteen ies sedAnther culture = When the pollen/anthers of the correct stage are collected and grown on suitable media, it is called as Tada meU ice = Itis used to produce haploid plants. = The selected flower buds are subjected to surface sterilisation with alcohol or hypochlorite solution for about 10-30 min. = They are rinsed repeatedly with sterile distilled water. = By using sterile forceps and a dissecting needle, the Elan ele Mele LCN CaS ce lea aaMi (oN (1m olele ee = The filaments must be removed prior to culture as they form callus at the cut ends. = The excised anthers may be cultured on solidified culture medium or placed on a filter paper bridge over a liquid medium. De aeThen is incubated at 25°C, in presence or absence of light. The plantlets are formed after 4-5 weeks of inoculation. Many plantlets are produced from single anther. These plantlets are carefully separated quite early and cultured on fresh root inducing medium containing agar to develop roots, thus forming entire plants. Alternatively the anthers are allowed to produce callus and from which shoots and roots respectively. haploid plantlot | mee 2 Anthers cultured ‘nutrient medium haploid plantleten Pollen on media eI Bceleaat-eelA) py aXetey ai arie t=} eloya) Root initiation Whole plant. Preteen ies sedFi lela ome imal aS Culturing of the embryo excised from the seeds on sterilised culture medium is called embryo culture. The selected fruit is surface sterilised for 15 min with alcohol and then washed thoroughly with distilled water. The fruit is cut and the seed or ovule is taken out with a sterile forceps and placed ona sterile petri dish. The seed or ovule is cut open and the embryo is separated. Bia -aco-Jo R101) ola oR Maan -reli-1c1\a (lace Roman -Malel dd -iale medium of culture vessel under aseptic conditions. The explanted embryos on culture vessels are kept at room temperature in diffused light. This resulting in the production of seedlings which are transferred to sterile growth promoting medium for further development. DesaMature embryos are usually selected as the very small globular embryos require a delicate balance of hormones. The dormancy period of seeds can be shortened. By the embryo culture it is possible to know the viability of seeds. By this culture it is possible to know the nutritional requirements of the aa has at various developmental stages. Used to propagate rare EH