Experiments on the preparation of plant chromosome specimens by the de-walled hypotonic method
Experiments on the preparation of plant chromosome specimens by the de-walled hypotonic method
Source: Laboratory Course in Genetics
Operation method
basic program
Materials and Instruments
Rye (Secalecereale), barley (Hordeumvulgare) seeds or onion (Alliumcepa) bulbs Move 1. Taking material: first soak the seeds for a number of hours, and then transferred to a petri dish padded with moist filter paper, placed in a constant temperature incubator at 25 ℃ germination, until the young roots grow to 1 ~ 2cm to take material; or bulb placed in a petri dish of water, placed in a constant temperature incubator at 25 ℃, until the root tip grows to 2cm or so to take the tip of the root (apical 0.5 ~ 1cm). 2. Pre-treatment: the root tip was placed in a penicillin bottle containing p-dichlorobenzene saturated aqueous solution, and immersed for 3～4h. 3. Pre-hypotrichosis: The root tips were treated in 0.075 mol/L KCl solution for 30 min. 4. Enzymatic de-wallization: Pour off the KCl solution and wash well with distilled water. Add mixed enzyme solution (cellulase and pectinase each accounted for 2.5%), enzymatic digestion at 25 ~ 30 ℃ for 2 ~ 3h. In the enzymatic process, it is best to gently shake the bottle, so as to promote the enzymatic reaction more fully. 5. Post-hypotonic: pour off the enzyme solution, rinse it slowly with distilled water for 2~3 times, and then leave it in distilled water for 10min for post-hypotonic treatment. 6. Fixation: methanol:glacial acetic acid (3:1) fixative for 30min. 7. Smear: take 2~3 root tips on a dry clean slide, cut off the tip 1~2mm, put 1~2 drops of methanol-glacial acetic acid (3:1) fixative on it, quickly crush the root tip tissue with forceps, spread it evenly on the slide, and swipe it over the flame of an alcohol lamp for 3~4 times. 8. Staining: Giemsa stock solution and 1/15 mol/L phosphate buffer (pH 7.2) mixed with 20:1, divided into staining cylinders, the dry preparation was placed in the staining solution, or the staining solution can be dropped directly on the slide, staining time of about 30 min. tap water rinsing slide, air drying can be microscopic examination. Observe the chromosomes under the low-power objective lens first, and then add microscope oil and convert it to oil lens for observation (if cedar oil is used, a coverslip is needed because the chromosomes will be discolored in the cedar oil) after a good mid-phase schizogony has been found. Selection of the better schizogony phases was photographed on a microscope equipped with a digital camera. For more product details, please visit Aladdin Scientific website.
Saturated solution of p-dichlorobenzene Methanol Glacial acetic acid 70% alcohol Cellulase Pectinase Giemsa stock solution Phosphate buffer (pH 6.8-7.2) KCl
Constant temperature incubator Microscope Slides Alcohol lamps