| One of the rewarding aspects of chromosome studies in plants is that
you get out into nature first. When botanists started to identify plant
groups and publish that information, chromosome number data were often
included. The technique was simple and inexpensive. Chromosome numbers
represent the first data set that leads to an understanding of the
genetics of any species. Obviously to study chromosomes you need
to remove the plant for further study. You need to be sensitive
about this. Are you on private property? Is the plant endangered and rare?
Are you going to disturb the habitat by removing the plant? These are
some of the issues which you need to be sensitive to before actually
removing a plant species for study. |
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| Identification of the plant species is critical. You need to consult
whatever keys you have at your disposal to be sure you have correctly
identified the specimen. In Virginia we use the Flora of West Virginia
by Strausbaugh and Core. We also use the Flora of the Carolinas
by
Radford, et. al. The species I am determining here was false Solomon's
seal, otherwise known as Smilcinia racemosa (Liliaceae). It is a very
common species and spreads easily from rootstock. Removing it for study
then is no problem. There were other members of the species in the area,
and the property I was on was public, not private. You still need to be
careful about what you do in public property. Just be careful and use
common sense when it comes to removing plants for study. |
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| The most important step in the process of study is to take a herbarium
specimen. Plants are collected and pressed between blotter paper. After
they are dried in the press they are mounted on a standard herbarium
sheet for preservation. This is the voucher specimen I use to cite what
I did in identification and chromosome number determination. Readers of
the article then can contact the Herbarium where these are stored for
further study. They can verify the correct identification of the
population I chose to work with. Notice the clay pot nearby. There is
still enough of the plant specimen to dig up and take into a greenhouse
environment. Transplantation will stimulate root growth. It is from the
roots that you obtain the chromosomes. |
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| It takes about two weeks for new roots to grow in a potted plant. The
fixative is a mixture of 3 parts Ethyl Alcohol to one part Glacial
Acetic Acid. You remove the plant from the pot and look for good thick
healthy roots. They are usually quite translucent and very visible. Use
forceps to take out the growing end of the root. The tips can sit in the
fixative up to 24 hours. If you cannot study the root tip within that
period of time, place them into a solution of 70% ethyl alcohol. Rinse
them in the alcohol a couple of times. You can store these tips in a refrigerator. I have obtained good chromosome stains from root tips
kept in alcohol for up to two years. You do need to change the alcohol
solution from time to time, however. |
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| The root tip is taken out of the fixative or storage alcohol and
placed on a micro slide. Add a drop of 2% acetoorcein onto the tip. This
is a mixture of 2 grams of orcein stain in 100 ml. of Glacial Acetic
Acid. You slowly warm this mixture in a beaker which is capped with a
funnel. The funnel allows the mixture to reflux, which means that the
molecules of the mixture will condense on the glass and move back down
into the liquid. This slow mixing insures that the orcein will liquefy in the acid. Filter this mixture. Next, make a solution of 9 parts of
this stain to 1 part 1 Normal Hydrochloric acid. The purpose of the acid
is that it will help to soften the tissue. The tissue comes directly
from the 70% alcohol onto the slide for staining. |
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| The method employed in this kind of work is called
the Squash Technique. You need to obtain a good thin cover slip. You add a small
amount of Mayer's Albumen onto the cover slip. This special solution can
be purchased as is from Carolina Biological
Supply. Smear the albumen
onto the cover slip. Make sure it is a thin smear. Then, using forceps,
place that cover slip over an alcohol flame in order to dry it. Leave it
as is while you then use forceps to macerate the tissue which is in the
stain. Place the slide with the macerated tissue over an open alcohol
flame. As you can see in the picture you just hold the slide. Warm
gently. This causes the stain and acid to work on the cells of the
tissue. They take the stain and become soft. |
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| The most important step in this process is shown on the right. Place
the micro slide onto a piece of paper towel. You place the cover slip albumen side down over the stained tissue on the
micro slide. Bring the
end of the paper towel over the cover slip and squash down as hard as you
can with your thumb. The towel removes the excess stain. The squashing
causes the tissue to be further macerated. The cells are pushed out into
smaller groups, some of them actually by themselves. If the cells are in
mitosis, then you can take a moment to examine the slide under a
microscope to see if chromosomes are present for study. If they are, you
can then make the slide permanent. |
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| To make the slide permanent you separate the slide from the cover slip
in a square Coplin Jar. The cells will be stuck to the cover slip, not
the slide. Take the cover slip through these mixtures: 1 part Ethyl
Alcohol to 1 part Glacial Acetic Acid; 3 pts alcohol to 1 pt acid; 9 pts
alcohol to acid; then two changes of 95% alcohol. This is a total of
five mixtures all in small Wheaton Dishes which handle just the cover slip. The purpose of this is to remove the water from the tissue,
as well as all of the acetic acid. This is not totally possible because
the final alcohol mixture has some water in it. But, two changes of that
final mixture will get rid of all the acid. If the acid remains in the
final preparation, the chromosomes will lose their stainability. |
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| The final process of making the slide permanent is to take a cleaned
micro slide and add to it a drop of mountant called Euparal. This is a
special mountant which is recommended for chromosome work. Most
microslides are preserved in a mountant called Canada Balsam. The ph of
this mountant is not good for preserving chromosomes. I have slides
which I made in 1965 that were mounted in Euparal and they are still
usable. Take the cover slip out of the last mixture of 95% alcohol and
place it, tissue side down, over the Euparal. Use forceps to carefully
lower the cover slip over the slide. This eliminates air bubbles. Let it
sit for 24 hours. Store the slides flat in a slide storage cabinet.
Click here to see the technique step by step. Also included are the
formulas to make the various solutions. |
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| I was awarded a grant from the National Science Foundation in 1981 to
purchase a good research microscope for chromosome studies. As you can
see, this microscope is quite ordinary in appearance. It is equipped
with phase contrast and has special lenses which are used in chromosome
study. The most important attachment is a drawing
tube. It is set up so
that you can see the chromosomes and also your hand with a pencil. You
then just trace over the chromosomes. My experience is as follows: find
a good metaphase or anaphase view of the chromosomes and draw them as
best you can. Make sure you are getting all of them and can determine
them individually. This drawing becomes what you count. You ought to
make several slides from different root tips to see if you are getting
the same number. You also should, if possible, repeat this process from
at least two more specimens from the original population to make sure
this chromosome number is the correct one. You don't want to publish
mistakes. |
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| This is an example of a drawing I made of the chromosomes of
Bermudiana,
the unofficial national flower of Bermuda.
This drawing was made of the chromosomes pairing in Prophase I of
meiosis. There are 32 pairs visible here, which means that the
chromosome number for this taxon is 2n = 64. The round dotted
circle is the nucleolus. The border is of the cell membrane.
The nuclear membrane disappears during prophase I of
meiosis. You can count them easily yourself, but notice there
are two pairs touching each other on the left edge of the
nucleolus. For other examples of what data come out of these kinds
of drawings, see my research work with Trillium. |
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