za Objective. To prepare temporary acetocar Ga parious stages of mitosis. ” RE! } me! slid anylatcohol, acetic acid, hydrochloric acid, ne stained mount of onion root tip to study QuiREMENTS Ibs, conical flasks or Onion bulbs, conical flasks/glass bottles, corked vial/tube, petridishes, scissors, f ceps, need] spel , scissors, forceps, needles, : acetocarmine, di : i resi, blotting paper ete ‘ine, distilled water, spirit lamp, microscope, proceDURE 1 Take a medium sized bulb of onion and trim off the old me roots from its base by means of asharp Place the onion on a conical flask/glass bottle full of water, with its base touching the water. Keep it for a week to grow the roots, Cut 5 mm off the tips of roots and put them into a vial containing a mixture of 1: 3 acetic acid and methanol. Keep for one hour. This process is called fixation. (Cutting of root tips should be done in the morning between 7.00 a.m. to 8.00 a.m. during the summer and between 9.30 a.m. to 11.30 a.m. during the winter). Remove 2 or 3 root tips and hydrolyse them by warming to 60°C in 1 N hydrochloric acid for 15 minutes. Fig. 6.1. Method of growing onion root tips. Remove the root tips and wash them thoroughly in water. Place a drop of acetocarmine on aside, Put one hydrolysed root tipin adrop and placea coverslip on the root. / Gently squash the root by tapping the coverslip with the blunt end of a pencil or needle until the cells separate and spread out into a very thin layer. Make sure that there are no air bubbles under the coverslip. Gently warm the slide over a flame for a few seconds. Observe first under the low power of the microscope to locate the dividing cells. Examine the different stages of mitosis under the high power of the microscope.“ty OBSERVATIONS Under low power of the microscope, rectang high power of the microscope following stag ith pink mu n scattered, Up, inet : ( ih Prophaso Anaphase Interphase 6.2. Different stages of mitosis in the onion root tip. 1. Interphase @ Itis a non-dividing phase of the cell cycle between two successive cell divisions. (i) Chromatin fibres appear in the form of a network within the nucleus. (iii) Nuclear envelope and nucleolus are distinct. 2. Prophase (Chromatin material shortens and condenses into thread like structures called chromosoma (i) Each chromosome consists of two chromatids, jointed at a point called centromere. (ii) Nuclear membrane and nucleolus start disintegration and disappear at the end of prophas 3. Metaphase @ A bipolar spindle develops in the cell. Chromosomes become thick and two chromatids each chromosome become clear. (i) Chromosomes become arranged at the equator of the spindle. (iii) Each chromosome get attached to the spindle fibres at its centromere. 4. Anaphase (®) ‘The two sister chromatids of each chromosome separate from the centromere and mot towards the opposite poles. (ii) The daughter chromosomes (separated chromatids) appear V, J, L and I shapes, dependt upon the position of centromere. 5. Telophase (i ‘The spindle disappears and the daughter chromosomes uncoil to form chromatin fibres! the two poles, (ii) Nuclear membrane and nucleolus reappears and two daughter nuclei appear at oppos! poles. Gif) Cytokinesis occurs by cell plate formation between the two daughter nuclei.Ap Nuclear membrane Chromatin fibros Nucloolus: |— Coll membrane Interphase stage Cell wall Nuclear Nuclear membrane [membrane Disappearing V3) _| Nucleolus nucleolus Chromosomes i: coromosomes |__ Cell wall Early prophase ' Daughter Re. pone A. |_- chromosomes fibres a. SN Chromatids A vi . YY | __spindle fibres Daughter cells Cell wall Chromosomes. :—— Daughter nuclei (Chromatids) Coll plate Nuclear Spindle fibres. membrane ; Late anaphase Telophase stage Fig. 6.3. Various stages of mitosis in onion root tip cells. PRECAUTIONS 1. The base of the onion bulb should be in contact of water while growing the roots. 2. Root tips should be fixed in the morning between 8 to 10 A.M. Pe 3. The slide should be warmed gently much above the flame of the spirit lamp.