Caitlin Marie's Article  *

“Polyploidization of Evergreen Azaleas” *

Caitlin Klimavicz

Introduction

              The characteristics that separate polyploid azaleas from diploid azaleas is the tendency of polyploids to have; thick leaves and petals, dark green foliage, blight resistant flowers, long lasting flowers, increased opportunity to cross with deciduous azaleas, and a better marketability due to the fact that they look healthier year round (ASA Journal, Summer, 2004: p.32).  The purpose of this project, spanning over two years, was to determine a reliable and easily repeatable way to produce polyploidy azaleas.     
              Diploid azaleas have 26 chromosomes (13 pairs).  The greatest number of chromosomes ever recorded was 152 (76 pairs) (Galle, 1987).  Some well known polyploids include: Haro-No-Sono, Wako, Taihei, and Getsutoku.  There are two types of polyploid azaleas, allopolyploids and autopolyploids.  Autopolyploids are usually sterile because of the odd number of chromosomes, while allopolyploids are usually fertile (Griesbach, “Polyploidy in Orchid Improvement”).  
             I have worked with my father for many years on a hybridization program aimed at developing sturdier azalea varieties that are better able to survive neglect and still look good.  Specific hybridization objectives include a plant that is vigorous, disease and insect resistant, floriferous, and has a flower that is long lasting.  Polyploidization should help us reach many of our goals.   Prior to my work on this project, our efforts to produce polyploid azaleas were limited to cross-pollenization, and the results were relatively poor.
            Azalea hybridizers sometimes wonder why a specific cross results in significantly lower germination rates when compared to other crosses.  Early in this project, I designed an experiment to prove that when seeds have an abnormal number of chromosomes then germination is extremely limited.    This phenomenology appears to be directly related to whether one of the parent plants was a polyploid azalea. 
          In an attempt to find an easily repeatable method with corresponding higher germination rates, to produce polyploid azaleas, radiation was administered to the seeds before germination.  Radiation is just one experimental technique to induce polyploidy.  Right now there are three ways of producing polyploid azaleas: to cross-pollinate, to treat the plant with colchicines chemicals, or to expose the seeds to radiation.  The most successful to date has been treatment of the plant with Colchicines.  However, this technique is difficult in application and the results are often inconsistent. (Galle, 1987)

Procedure

          Seeds from known diploid and polyploid azaleas were collected and tested to determine if more diploid azalea seeds and x-rayed azalea seeds would germinate than polyploid azalea seeds.  They were divided into three groups, each weighing 1.7 grams.  The three groups of seeds were divided into two diploid groups and one polyploid group.  One diploid group was X-rayed ten times (total of one second) at 15.0 mVs.  The X-ray machine, 123 kVp, set 41 cm away from the envelope. 
          The three groups of seeds were each planted in a separate container.  The containers were placed under grow lights and watered.  After the seeds germinated the number of seedlings in each container were counted. 
           The following year, stomata measurements were taken to determine if the seeds that were x-rayed are polyploids.  To measure the stomata of the azaleas, a microscope was calibrated using a micro ruler and the lines built into the eyepiece.  Then leaves were collected from the plants to test.  The leaves were labeled with a permanent marker on the topside of the leaves.  The underside of the leaf was painted with clear nail polish.  When the nail polish dried, it was peeled off the leaf.  The clear piece of nail polish was placed on a clean slide with a drop of water.  A cover slip was placed on top.  Excess water was squeezed from under the cover slip, and the slide was labeled with a permanent marker.  The slide was placed under the microscope and the microscope was focused on the leaves’ stomata.  The lines in the eyepiece that were calibrated earlier were used to measure the length and the width of three different stomata on each leaf.

Results

          The number of seedlings in the diploid container that was not x-rayed was 264.  The polyploid container had only one seed germinate.  The x-rayed container had nine seeds that germinated.  Seeds with an abnormal number of chromosomes do not germinate as readily as normal diploid seeds, therefore making polyploid azaleas more difficult to hybridize. The experiment determined that loss of germination was directly related to whether one of the parent plants was a polyploid azalea.  There was also loss of germination in the x-rayed seeds, yet not too as great an extent. 

         The next phase of the project was to determine if the x-rayed azaleas were actually polyploid.  Stomata measurements can be used as a means for determining if an azalea is a polyploid.  Based upon the two major groups of points, it appears that the x-rayed azaleas are polyploids.  A t-test was performed to determine if the two populations were statistically different.  The test confirmed that they are different populations. 
          Azaleas normally have two nuclear organizers.  This picture clearly shows the four nuclear organizers present in the nucleus of an x-rayed azalea root tip cell.  This is also strong evidence that there are more chromosomes than normal in this cell, i.e. polyploid (Griesbach).     

  Discussion

           Using standard azalea crossing techniques as a means to produce polyploid azaleas does not produce acceptable levels of germination.  As determined in the first phase of this project, the germination rates for a polyploid cross is significantly lower than normal diploid x diploid crosses.
           The hypothesis in x-raying azalea seeds is that this is a reliable and easily repeatable method for producing polyploid seedlings.    But we have to have some way to determine if we actually produced a polyploid azalea.  The best way to measure polyploidy is to count the chromosomes under a microscope.  This technique did not work in this case because it was too late in the year to get active root tips.  The polyploidy in this case was taken into account by using stomata measurements.  This is where the length and the width of stomata on each leaf are measured to determine the lengths to widths ratio that should vary between diploid and polyploid azaleas.  Another technique that could have been used was FLOW.  FLOW is a system that measures the amount of genetic material in a sample and not how many chromosomes; therefore it is not very effective in determining polyploidy (Griesbach). 
          The cross-pollinated polyploid seeds were tested to determine if in fact there were embryos inside the seeds or if no embryos was causing the lack of germination.  To accomplish this test, seed were soaked in Carmen dye for several minutes.  If they floated it was a sign that there might not be an embryo inside.  Then once the dye soaked in to the seeds, they could be looked at under a microscope to see the embryo.  It was determined, using this method, that most of the seeds from the polyploid cross did contain embryos.  This leads us to believe that the cause of decreased germination was not lack of embryos (Griesbach).   
           This project is not perfect and there were two main places where error could have occurred.  The first was in the measurements that were taken of the stomata.  These measurements were done on a very small scale so it would not have been hard to make an error.  That is why three measurements were taken from every leaf.  The second possible point of error is that even though the stomata measurements are designed to reveal the polyploidy of the azaleas it is not the same as actually counting the chromosomes and therefore even if the measurements are performed correctly it is not an absolute measure of polyploidy. 
          Future experiments could include, actually counting the chromosomes, or testing the other methods, such as the chemical treatments applied when the plant is full grown. 

Conclusion

Even though the data seems to suggest that radiation can produce polyploid azaleas, the question this project initially set out to answer definitely remains uncertain.  The initial question was, “Is there a reliable and easily repeatable way to generate polyploid azaleas?”  The answer given by the stomata measurements concludes that radiation is a reliable way to generate polyploid azaleas.  Yet on the other hand, without an actual chromosome count one cannot be certain that the x-rayed seeds were actually polyploid.  The germination rate for the x-rayed seeds was still very low compared to diploid seeds, and the radiation absorbed by the seeds could cause other problems down the line that have not been observed yet in this stage of growth.  This reduced germination rate also impacts the ability of this method to easily generate new and exciting polyploid azaleas.

Acknowledgements

          I would like to thank Dr. Robert Griesbach, USDA, for his help with the measuring of the chromosome count and looking at the seeds for embryos.  I would also like to thank him for his many ideas and suggestions that have improved my project.  I would also like to thank Dr. John Klimavicz of Ashburn Farm Animal Hospital for the use of their x-ray machine.

References

Eeckhaut, Tom G.R., et al. “Occurrence of Polyploidy in Rhododendron luteum sweet, Hardy Ghent, and Rustica Hybrids.” Azalean 26.2 (Summer 2004): 32‑37. Galle, Fred C. Azaleas. Portland, OR: Timber Press, 1987.
Griesbach, Rob, Ph.D. Personal interview. 4 Sept. 2004.
Griesbach, Rob J. “Polyploidy in Orchid Improvement.” American Orchid Society Bulletin 54.12 (Dec. 1985): 45‑58.
Griesbach, Rob J., PhD. “Rhododendron Flower Color Genetic/ Cultural Interactions.” Rhododendron Society Bulletin 12.2 (1986): 20‑21 Miller, Kenneth R., and Joseph Levine. Biology. New Jersey: Prentice Hall, 1995

Bio

Caitlin Klimavicz is currently (2006) a junior at James Madison HS in Vienna, Virginia.  She has regularly attended azalea meetings with the Northern Virginia Chapter since 1989.  She has won first place two years in a row at her high school science fair, and received a first and second place award in the regional science fair based on this project.  

* Reprinted by permission of the author and the Azalea Society of America, which previously published the article in its quarterly journal The Azalean (Fall 2005, Vol 27(No. 3):58-61).

*for other pictures and scedules used in this article see Abum 4