Rychlá cytologická metoda – studium fází mitózy Roztlakové preparáty Materiál: kořenové špičky rostlin Definice meristému •v raném vývoji embrya se dělí všechny buňky •později je dělení buněk omezeno pouze na malé oblasti, ve kterých si buňky zachovávají embryogenní charakter a schopnost dělení (apikální meristémy = primární meristémy – kořene, prýtu) - jejich činností vznikají nové buňky, které po diferenciaci vytvářejí rostlinné tělo •sekundární meristémy –kambium a felogén –axilární meristémy WHAT IS A MERISTEM? In the early stages of development of the plant embryo all the cells undergo division, but with further growth and development cell division and multiplication become restricted to special parts of the plant which exhibit very little differentiation and in which tissues remain embryonic in character and the cells retain the ability to divide. These embryonic tissues in the mature plant body are called meristems. Charakteristika meristematických buněk •malé, izodiametrické buňky •tenká primární buněčná stěna •velké jádro (jádro-plazmový poměr) •velká hustota protoplastu – malé vakuoly • • Buněčný cyklus G0 fáze: buňka se nedělí, je součástí pletiva G1 fáze (z ang. gap = mezera): na buňce není pozorovatelné co se v ní děje, probíhá syntéza RNA a bílkovin S (syntetická) fáze: syntéza DNA G2 fáze: syntéza bílkovin pro vznik a dělení jádra (histony a mikrotubuly) M fáze (mitóza): nepřímé dělení jádra C fáze (cytokineze): dělení celé buňky I (interfáze): doba, kdy se buňka připravuje na dělení, tj. G1, S a G2 fáze mujweb.cz/Veda/biologie/b.%20cyklus.htm. Mitóza Mitosis | BioNinja Podélný řez apexem stonku Coleus příklad stavby a lokalizace apikálního meristému prýtu a axilárního meristému (Coleus_SAM) http://www.esb.utexas.edu/mauseth/weblab/webchap6apmer/6.1-1.htm Longitudinal section of shoot tip of coleus (Coleus). The central vertical structure is the shoot tip, which ends in the shoot apical meristem (the reddish structure with two horn-like leaf primordia). On either side of the apical meristem are two larger leaves that extend up out of the top of the micrograph. In the lower third of the micrograph are two axillary buds, each with two small leaf primordia. Coleus is a favorite plant for teaching about apical meristems and leaf production because it has decussate phyllotaxy (that is, each node has two opposite leaf primordia, the next node down also has two, but which are arranged at 90 degrees from the two above). We can see two young leaf primordia (the "horns" on the apical meristem), then two older ones, and finally two even older leaves that have the two axillary buds. Notice that there are bumps on the surface of the stem, above the two axillary buds -- those are the very edges of another leaf pair, with one leaf going back into the screen and the other coming up at us (both leaves have been cut off). In plants that have distichous phyllotaxy (with just two rows of leaves, as in irises), a median longitudinal section like this would pass through every leaf (or with a different orientation, it would miss every leaf). In plants with spiral phyllotaxy (just one leaf per node), a median longitudinal section often misses many leaves, or just barely passes through one of their edges. Apikální meristém kořene u lnu (Linum sp.) vlastní meristém klidové centrum (QC) kořenová čepička kolumela = sloupek, střední část kořenové čepičky Buňky apikálního meristému kořene hrachu setého (Pisum sativum) •přírodní •karmín - červec nopálový (acetokarmín) •orcein - lišejníky (Lecanora, Roccella) (laktopropionový orcein) •hematoxylin - dřevo kampešky (Haematoxylon campechianum) • •syntetická •nigrosin •krystalvioleť •Feulgenova nukleární reakce (s leukobazickým fuchsinem) Barviva pro barvení v rychlých cytologických metodách Karmín kyselina karmínová [USEMAP] Dactylopius coccus Cochineal, Natural Red 4, C.I. 75470 Carmine, also called Cochineal, Natural Red 4, or C.I. 75470, is a pigment of a bright red color obtained from the carminic acid produced by the cochineal insect, and is used as a general term for a particularly deep red color. Carmine is used in the manufacture of artificial flowers, watercolors, rouge, cosmetics, food additives and crimson ink, and in the painting of miniatures. Carmine may be prepared from cochineal, by boiling dried insects in water to extract the carminic acid and then treating the clear solution with alum, cream of tartar, stannous chloride, or potassium hydrogen oxalate; the coloring and animal matters present in the liquid are thus precipitated. Other methods are in use; sometimes egg white, fish glue, or gelatine are added before the precipitation. The quality of carmine is affected by the temperature and the degree of illumination during its preparation, sunlight being requisite for the production of a brilliant hue. It differs also according to the amount of alumina present in it. It is sometimes adulterated with cinnabar, starch and other materials; from these the carmine can be separated by dissolving it in ammonia. Good carmine should crumble readily between the fingers when dry. Carmine lake is a pigment obtained by adding freshly precipitated alumina to decoction of cochineal. Carmine can be used as a staining agent in microbiology, as a Best's carmine to stain glycogen, mucicarmine to stain acidic mucopolysaccharides, and carmalum to stain cell nuclei. In these applications, it is applied together with a mordant, usually an Al(III) salt. Orcein archil, orchil, lacmus, litmus a C.I. Natural Red 28, (E121) červené v kyselém pH, modré v alkalickém pH Orcein, also Archil, orchil, lacmus, litmus, and C.I. Natural Red 28 is a dye extracted from a lichen. It is red in acidic pH and blue in alkaline pH. Orcein is approved as a food dye, with E number E121. Its CAS number is 1400-62-0[1]. Its chemical formula is C[28]H[24]N[2]O[7]. It forms dark brown crystals. Orcinol is extracted from archil lichen, Rocella tinctoria. It is then converted to orcein by ammonia and air. Orcein is a reddish-brown dye, orchil is a purple-blue dye. Orcein is also used as a microscopical stain. It is a mixture of phenoxazone derivates - hydroxyorceins, aminoorceins, and aminoorceinimines. Mitotické jedy •halogenové deriváty aromatických uhlovodíků (p-dichlór benzén, -bróm naftalén, 8-hydroxychinolin) •alkaloidy (kolchicin, vincristin) •herbicidy (oryzalin) • Rostlinné alkaloidy a herbicidy •kolchicin •vincristin • • •oryzalin [USEMAP] Obecný postup • •fixace Carnoy, Farmer •macerace HCl •roztlačení •barvení acetokarmín, laktopropionový orcein •projasnění levulózový sirup, glycerol fixačně-macerační směs předpůsobení Fixačně-macerační směs Dostál J. (VÚOK Průhonice) •1 díl 96% etanolu •1 díl normálního butanolu •1 díl ledové kyseliny octové •1 díl konc. HCl (35 – 36%) •4 díly destilované vody Pracovní postup Laktopropionový orcein roztlakový preparát Allium cepa Olympus BX-51 procházející světlo Olympus BX-51 DIC