Root Tip Chromosomes

	CHROMOSOMES IN ROOT TIPS David B. Fankhauser, Ph.D., Professor of Biology and Chemistry University of Cincinnati Clermont College, Batavia OH 45103
Some of the best mitotic figures can be seen in onion root tips	This page has been accessed times since 11 January 2004. 7 October 1992, later revisions 31 Dec '95, 14 Jan. '97, 5 Jan '98	anaphase and metaphase from an onion root tip

See related protocols on Sprouting Seeds and Use of Oil Immersion Lens .

Chromosomes were first seen by C. Nägeli in 1842, and named in 1888 by W. Waldeyer. Walther Flemming studied and documented the behavior of chromosomes during cell division, a process he termed mitosis. We will perform experiments similar to these early scientists.

Cell division is especially rapid in the growing root tips of sprouting seeds. The chromosomes in dividing root tip cells can be demonstrated if, after we sprouting seeds or bulbs, we harvest the young root tips, and then fix, acid digest, stain, squash, and view them under a microscope. The impressive chromosomes viewed in our lab , best to least, have been:
onion I, onion II, wheat , rye , lentils , barley , alfalfa , broccoli , mung and soy beans.

Here is the general plan of the procedure, supplies and equipment needed:

EXPERIMENTAL PLAN SUPPLIES: EQUIPMENT:

Carnoy's fixative (1:3 HOAc + EtOH)

1N HCl

Feulgen stain

45% HOAc

Freshly sprouted seeds , about 2-3 cm long.

(rye, wheat, lentils, alfalfa, onion, etc)

Wasserman tubes (13 x 100 mm) with corks

Pasteur pipets

Constant temperature "hot block", 60^oC

microscope slides

razor blade and/or fine scissors

cover slips

THE DAY PREVIOUS:

1. SET UP: Fill out a label with sprout type, seat number, initials and the date, apply to a clean Wasserman tube.

2. HARVEST AND FIX: Cut off the last 6 mm (1/4th inch) of root tip from freshly sprouted seeds (no longer). Using a long tipped Pasteur pipet, place 5 of them in the labeled test tukbe. Add 2 mL Carnoy's fixative, ensure all tips are immersed. Securely cork so that it is air tight. Fix the root tips in Carnoy's fixative for at least 24 hours.

THE NEXT DAY: DIGEST, STAIN AND SQUASH THE FIXED ROOT TIPS:

3. SET UP: Label an empty Wasserman tube with your seat number and the type of root tip. Remove two fixed root tips with a Pasteur pipet, transfer to the test tube.

4. ADD HCl: Add 1-2 mL of 1N HCl with a Pasteur pipet to the root tips.

5. DIGEST AT 60 C. Here, five sets of root tips are ready for digestion. Incubate the root tips in 1N HCl at 60C for 12 minutes in a constant temperature "hot block."

6.REMOVE THE HCl with Pasteur pipet, discard in drain with running cold tap water.

7. ADD FEULGEN STAIN: Add 1 mL Feulgen stain (caution: this stain does not look brightly colored, but stains strongly. Do not get on clothes, fingers, books, etc.)

8. STAIN FOR 10 MINUTES: Let sit at R.T. for 10 minutes until the very tip of the root shows distinct dark coloring.
SETUP SLIDE: Put a drop of 45% HOAc on a clean microscope slide.
Remove the Feulgen stain with a Pasteur pipet, discard into the drain.

9.ADD TIP, TRIM: Place the soft root tip in the HOAc on the slide. With a scalpel or razor blade, remove all but the red-stained very tip of the root.

10. ADD COVER SLIP, note that the tip will spread out somewhat.

11. SPREAD CELLS TO MONOLAYER: Place the assembly on a piece of white paper and tap gently and straight down with the eraser of a pencil until the stained tip is spread out to a faint purple monolayer. Do not smear side-wise, it shears the chromosomes.

12. Examine under the microscope at low power to ensure that the cells are adequately spread to a monolayer. If so, examine under higher power. Locate mitotic figures (near the tip end), and switch to oil immersion (1000x).

Here are some nice images of chromosomes from onion root tip, taken January 2005.

13. Illustrate examples of each mitotic stage (pro-, meta-, ana- and telophase). Prepare a second squash with a different species, and illustrate its mitotic stages, noting any differences observed between the two species.

Here is another page of pictures of root tip chromosomes in recent labs.

These are the four classic stages of mitosis:

prophase metaphase anaphase telophase (left) (& metaphase)

14. Show chromosomes of two species of plants you have prepared to the instructor for 5 points each.

EXPERIMENTAL PLAN	SUPPLIES:	EQUIPMENT:
	Carnoy's fixative (1:3 HOAc + EtOH) 1N HCl Feulgen stain 45% HOAc Freshly sprouted seeds , about 2-3 cm long. (rye, wheat, lentils, alfalfa, onion, etc)	Wasserman tubes (13 x 100 mm) with corks Pasteur pipets Constant temperature "hot block", 60^oC microscope slides razor blade and/or fine scissors cover slips

	1. SET UP: Fill out a label with sprout type, seat number, initials and the date, apply to a clean Wasserman tube.
	2. HARVEST AND FIX: Cut off the last 6 mm (1/4th inch) of root tip from freshly sprouted seeds (no longer). Using a long tipped Pasteur pipet, place 5 of them in the labeled test tukbe. Add 2 mL Carnoy's fixative, ensure all tips are immersed. Securely cork so that it is air tight. Fix the root tips in Carnoy's fixative for at least 24 hours.

	3. SET UP: Label an empty Wasserman tube with your seat number and the type of root tip. Remove two fixed root tips with a Pasteur pipet, transfer to the test tube.
	4. ADD HCl: Add 1-2 mL of 1N HCl with a Pasteur pipet to the root tips.
	5. DIGEST AT 60 C. Here, five sets of root tips are ready for digestion. Incubate the root tips in 1N HCl at 60C for 12 minutes in a constant temperature "hot block."
	6.REMOVE THE HCl with Pasteur pipet, discard in drain with running cold tap water.
	7. ADD FEULGEN STAIN: Add 1 mL Feulgen stain (caution: this stain does not look brightly colored, but stains strongly. Do not get on clothes, fingers, books, etc.)
	8. STAIN FOR 10 MINUTES: Let sit at R.T. for 10 minutes until the very tip of the root shows distinct dark coloring. SETUP SLIDE: Put a drop of 45% HOAc on a clean microscope slide. Remove the Feulgen stain with a Pasteur pipet, discard into the drain.
	9.ADD TIP, TRIM: Place the soft root tip in the HOAc on the slide. With a scalpel or razor blade, remove all but the red-stained very tip of the root.
	10. ADD COVER SLIP, note that the tip will spread out somewhat.
	11. SPREAD CELLS TO MONOLAYER: Place the assembly on a piece of white paper and tap gently and straight down with the eraser of a pencil until the stained tip is spread out to a faint purple monolayer. Do not smear side-wise, it shears the chromosomes.

CHROMOSOMES IN ROOT TIPS