Written By: Rob Shepherd
Last Updated: 08.20.2010
The goal of this document is to provide a protocol in near laymen’s terms with the purpose of determining the ploidy of Phalaenopsis hybrids and seedlings that have been treated with colchicine to induce polyploidy.
This document is a work in progress as I go through the process of learning how to do Phalaenopsis chromosome counts using the Root Tip Squash Technique. As I go along I’m trying to add little helpful hints and a few bits of humor to carry you along my little journey and hopefully help you avoid some of the trouble I ran into. Please come into this with lots of patience, some common sense, knowledge of lab safety and hopefully a reasonable biology and/or botany background. Don’t be afraid of making mistakes and be prepared to practice!
As soon as I have some time I'll start getting photo's posted and some more resources for equipement.
Half the challenge of this entire process is getting the lab materials. They are expensive and the chemicals are very hazardous. If you can not have hazardous chemicals shipped to your work, make sure you can find a local source that will allow you to pick them up. Otherwise you’re going to have a very hard time getting your supplies. None of the lab supply houses will ship these chemicals to a home address.
- Binocular or Trinocular Scope w/ 20X, 40X and 100X oil immersion lens (You will need a binocular scope if you want to use a digital microscope camera.)
- Binocular or Trinocular Dissecting Scope
- Green Filter
- Slides w/ Cover Slips
- Vials w/ Stoppers
- Alcohol Lamp
- Tweezers and dissecting needles, a very small hypodermic needle is ideal to have.
- Wax Pencil or permanent marker that will write on glass.
- Orcein stain and/or Giemsa and Hemetoxilin stain
- Glacial Acetic acid
- Ethyl Alcohol (95%)
- Concentrated Hydrochloric acid
- Gum Mastic
- Immersion Oil
- Blotting Paper
Computer Equipment (Not required but highly recommended)
- USB Digital Microscope Camera (Higher Mega pixels the better, I would suggest 5MP of higher)
- Computer running Windows 98, XP or higher to run the Camera Capture software
- Photoshop or other image manipulation program
Chemicals and Reagents
8-Hydroxyquinoline Solution Preparation:
Pretreatment of root tips with 8-Hydroxyquinoline will cause contraction and improve spreading of chromosomes. Prepare a 0.002M solution of 8-Hydroxyquinoline (molecular weight = 145.15) by dissolving 0.029g of 8-Hydroxyquinoline in 100ml of distilled water. (reference 2.)
A small amount of ethanol may be needed to get the 8-Hydroxyquinoline to go into solution. Please note, do not use denatured ethanol, the denaturing chemicals can interfere with the final quality of the sample. Everclear is a reasonable alternative to lab grade ethanol.
1 Normal HCL Solution Preparation:
A concentration prepared with a graduated cylinder is the preferred method. Table 2 shows the amounts of HCl stock solutions of different concentrations required to prepare 1-liter of 1 M, 3 M, and 6 M HCl. A lesser volume of HCl field solution can be prepared by proportionally reducing the volume of HCl stock solution. For example, to prepare 250 mL of 1 M HCl from 30% muriatic acid, reduce the volume of HCl stock solution by 1000 mL/250 mL, a factor of 4: 106 mL/4 = 27 mL (30% HCl), adding enough distilled water to achieve the required volume of 250 mL.
Table 2. Volume of HCl stock solution required to prepare 1 liter
HCl stock solution concentration, wt %
Volume of HCl stock solution required, (mL)
1 to 2% Aceto Orcein Stain Preparation:
- 100ml 45% acetic acid
- 1 to 2 grams orcein
- Glass vial for stain storage
After not liking the results of the recommended 1% solution, I decided to change to a 2% solution. This is something you may have play around with to get the right amount, it may vary depending on the quality of the orcein. Prepare the 45% acetic acid by pouring 45ml glacial acetic acid into 55ml of distilled water. Since you will be using 45% acetic acid on a frequent basis during the entire process, I recommend preparing and storing a 500ml batch of 45% acetic acid for future use.
Place the 2 grams of orcein in the 45% acetic acid and heat to dissolve the orcein. Cool the solution and filter to remove any undissolved stain. Store the stain in the glass vial for future use.
TIP: Orcein will stain anything including your glassware and plastics. I found that OxyClean completely neutralizes the dye and will even remove it from white plastic.
TIP: I also found out the hard way that 45% acetic acid will quickly eat through rubber. Do NOT store the aceto orcein stain in a dropper bottle with a rubber bulb. The bulb will quickly disintegrate and you’ll have one hell of a mess. Even those nice lab grade dropper bottles will not work
Root Tip Counting Protocol:
- Sever active root tips, about 10mm long, and place in a small vial containing 0.002 M 8-hydroxyquinoline solution for 3 to 5 hours at about 65ºF (18ºC). Note: when you start looking at chromosomes under the scope, if you are not getting good contraction and/or you are having issues with getting them to spread out in the first place to tweak. You may need to increase the strength of the solution, increase the temp a little or just let the root tips soak a little longer.
- Wash with water.
- Fix with 2:1:1 mixture of 95% ethanol, chloroform, and glacial acetic acid for at least 10 minutes at about 50ºF.
- Hydrolyze with 1N HCl at 140ºF (60ºC) for 3 to 5 minutes.
- Wash with water.
- Place in 45% acetic acid for 10 minutes. (It may also be stored for several days in the refrigerator.)
- Transfer the root to a clean slide and cover with 45% acetic acid to keep roots from drying.
- Remove root cap under a dissecting microscope.
- Select clumps of cells that were underneath the root cap. Depending on where the root was growing, the active cells can be in different areas. In many cases the area of cells close to where the root cap connects with the base of the root is a very active area.
- Separate cells with needles. The thing I really didn’t understand here was that you REALLY want to separate out the cells. Take little clumps of cells and move them to other slides putting a drop of 45% acetic acid on the slides to keep them moist. Then under the scope, separate out the cells as much as possible so they are no longer a clump. Your goal is to end up with one layer of cells so that when you squash the cells they are not on top of each other.
- Blot out acetic acid, add 1 to 2 drops of aceto-orcein stain, and place slide in a chamber saturated with 45% acetic acid for 10 to 30 minutes. (I’m having to do at least 30 minutes to get good staining.) The slide should be placed on a pedestal of some kind to keep it above the acetic acid. I've been using glass petri dishes for the pedestal and had found a square glass container with a silicon seal (from Ikea) that has served me well. The slide is placed in the chamber to prevent the stain from evaporating and the plant material from drying out.
- Place cover glass, tap cover with the point of a needle or slide tapper, and remove excess stain.
- Place bibulous paper over slide and press firmly with thumb to flatten cells and remove excess stain.
- Seal edges of cover glass with paraffin-gum mastic mixture or dental wax.
- Count chromosomes with a compound microscope under an oil immersion lens. (reference 2.)
Tips for Counting Chromosomes on the Computer
The advent of digital cameras has enabled a whole new process for counting chromosomes. It is significantly much easier to do a count on the computer for beginners than it is to do the count looking through a microscope. You also have the advantage of a very permanent record of the count that can be referenced and analyzed at any point. I found that working off a live video feed on a monitor allows me to quickly find cells with good chromosomes and then I can take photos from the computer at different focal points on the cells I'm going to count. This allows you to get all the chromosomes in focus across a range of photos. It's almost impossible to get all of the chromosomes in focus in one photo, especially for beginners that are still working on their technique.
Special Thanks to Dr. Dean Stock for his guidance and support through this process. Without his experience and help along the way, it would not have been possible for me to gain the ability to do counts. I'm sure his efforts to educate all of us and train those that were interested will benefit the orchid community for many generations to come as a new wave of responsible hybridizers fill this world with orchid hybrids that are not only beautiful, but have clean ploidy so that future generations can continue to breed with them.
1.) “Assessing… Dilute HCL Solution” http://soils.usda.gov/technical/technotes/note5.html
2.) Tanaka, Ryuso and Kamemoto, Haruyuki, 1984. Orchid Biology Reviews and Perspectives, III, Comstock Publishing Associates, Ithaca and London. p: 328-330.