Questions and Answers
August E. Kehr
Geneticist, Plant Science Research Division, Agricultural Research Service
U. S. Department of Agriculture, Plant Industry Station, Beltsville, Maryland
Questions on colchicine technique, tetraploids, and their nature are continually being raised by persons interested in the genus Rhododendron. It is hoped that the following questions and answers will be helpful in the furtherance of this fascinating subject.
Q: What are the characteristics of a tetraploid derived by doubling the chromosome number of a plant?
In comparison to diploids (plants with two sets of chromosomes - one set from each parent), tetraploids generally:
- Have larger, heavier textured flowers which last longer
- Have cells with twice the volume or about 1¼ times diameter. Cell size is most readily seen in pollen and stomate cells.
- Have lower length-width ratio of of leaves (i.e., leaves are wider)
- Have heavier textured leaves and thicker stems
- Have four sets of chromosomes, two sets from each parent
Q: Do tetraploids help overcome sterility?
Yes and no. If the original diploid is fertile, the tetraploid derived from it may be less fertile. Such tetraploids have four sets of chromosomes that are all similar, and are known as autotetraploids. However, if the original diploid is sterile because it was developed from a hybrid of two very unlike species; as for example, an azaleodendron, the derived tetraploids is likely to be fertile, and highly so. Such tetraploids have four sets of chromosomes, only two of which are similar. Such tetraploids are known as allotetraploids.
Q: Do tetraploids have any advantage in making crosses?
A tetraploid derived from a sterile diploid will enable one to make a cross which cannot be made at the diploid level. This is particularly true of sterile hybrids between species. Other than such special cases, there is no particular advantage of tetraploids over fertile diploids in making crosses. With a few exceptions induction of tetraploidy does not usually overcome cross incompatibility.
Q: Can one cross plants of unlike chromosome number?
Yes, quite readily. In general, chromosome numbers are not in themselves a barrier to hybridization. Another general rule (to which there are sometimes exceptions - as in many biological rules) is that when plants with unlike chromosome numbers are mated, the cross will be more likely to succeed if the female or seed parent has the larger chromosome number. However, there are instances in which the reverse is true.
Q: What is colchicine?
Colchicine is a medicinal chemical extracted from either the seeds or corns of Colchicum autumnale L. (meadow saffron or fall crocus). It is an alkaloid, and highly poisonous if taken internally in any significant amount. It may also cause skin irritation or temporary blindness if it gets in the eye. Physicians have long used colchicine as a treatment for gout in humans; but the effect of doubling chromosomes in plants was discovered only in 1937. Colchicine is a yellow powder, highly soluble in water, and will keep for an indefinite period in powder form. Also, when dissolved in water, it will keep for long periods, or until (or if) a fungal or bacterial growth forms in the solution. In handling colchicine, extreme care should be taken to avoid swallowing it in any amounts, or getting it into the eyes.
Q: Where can I get colchicine?
Colchicine is not sold in most drug stores, and we are informed that the form used by physicians is not suitable for plant work. Only the U.S.P. form should be used. Individuals should form a group and order through a local physician or a University contact from a chemical supply house. In addition to chemical supply houses the following firms handle this chemical: Benson-Maclean, Bridgeton, Indiana 47836. S. P. Penick & Co., 100 Church Street, New York, New York 10007, phone (212) 267 -1970. Nutritional Biochemicals, 26201 Miles Road, Cleveland, Ohio 44128, phone (216) 662-0212. Centerchem Inc., Empire State Bldg., Suite 6208, 350 Fifth Avenue, New York, New York 10001, phone (212) 594-8520. J. H. Walker & Co., Inc., 22 West First Street, Mount Vernon, New York 10550, phone (914) 699-4840. Roussel Corporation, 155 East 44th Street, New York, New York 10017, phone (212) 697 - 5820
Q: What methods of chromosome doubling should I use?
From my experience with rhododendrons, treatment of germinating seeds appears to be the most successful method. I know of no one who has successfully induced tetraploids in rhododendrons by treatment of the growing tips. Azaleas, on the other hand, apparently are more readily doubled by treatment of the growing tips, and five tetraploid forms of azaleas have just been released to nurserymen by workers in our research unit. Briefly, the treatment of germinating seeds is done as follows:
- Germinate seed on absorbent filter paper or blotter paper moistened with water in a petri dish or similar covered container.
- When seed begins to germinate and small roots appear, add colchicine solution at .02 percent strength.
- Leave germinating seed in colchicine for 2 to 3 days.
- Remove and plant only seed that has germinated and been exposed to the colchicine. The un-germinated seed frequently germinates later and is not affected by the colchicine treatment.
- Carefully remove and discard (or plant elsewhere) all fast growing seedlings representing about 95 percent of the total population. These are not affected by the colchicine treatment. The affected seedlings usually have thickened and swollen roots and cotyledons, and are very slow in starting to grow. Unless the fast-growing, unaffected seedlings are removed, the affected seedlings are smothered out.
- The slow-growing, affected seedlings can be speeded up in their growth by additional artificial light for 18 hours per day.
Q: How dangerous is colchicine?
Colchicine is an alkaloid and is very poisonous when taken internally in any significant amount. However, there is no record of any fatalities or injuries from the use of this chemical in doubling plant chromosomes. In fact, it is less dangerous than nicotine, another alkaloid which is familiar to most persons. Finally, the concentrations at which colchicine is used for doubling chromosomes are extremely low, usually 0.02 percent, and hence are less dangerous than concentrated solutions or the pure chemical.
Q: Where can I get information on the colchicine technique?
For detailed information on colchicine and its application the book entitled "Colchicine," by O. J. Eigsti and P. Dustin, Jr., is the best reference. This book can be obtained in most large libraries. Other references are:
- "The Use of Colchicine in Plant Breeding," available from Office of Information, U.S. Department of Agriculture, Washington, D.C. 20005, or from August E. Kehr, Plant Industry Station, Beltsville, Maryland 20705. This publication is old, but still good.
- Colchicine - "Induced Tetraploid Azaleas," by Pryor and Frazier - found in HortScience, Volume 3 (4), Winter 1968, pages 283-286. Single copies of this journal may still be obtained for $2.50 ($1.50 for members of the Hort Society) from American Society for Horticultural Science, Post Office Box 109, Saint Joseph, Michigan 49085.
- "A Tetraploid Rhododendron carolinianum," by A. E. Kehr found in American Rhododendron Bulletin, Volume 25 (1), January 1971.
Note: The concentration of colchicine in this article should be corrected to read 0.25 percent.
Q: What happens if one doubles a tetraploid?
When the number of sets of similar chromosomes reaches eight or more there is usually a reduction in plant vigor. If the sets are unlike, however, relatively higher chromosome numbers can be built up without a significant decrease in plant vigor. The highest chromosome number reported in Rhododendrons is in the species R. manipurense with 156 chromosomes, a duodecaploid with six times the diploid number of 26. Despite the large number of chromosomes, this species is very vigorous.
Q: Why have I failed to succeed in doubling chromosome sets with colchicine?
Colchicine is effective only in cells which are actively dividing, and has no effect whatsoever on resting cells. Likewise, in growing tips the target is a few cells, or at best an incredibly small area. Thus for success the colchicine must penetrate through the bud tissues to reach this area and the target cells must be actively dividing. Unless these two conditions are met, there can be no successful treatment. Likewise, in seedling treatment one must be careful to remove fast-growing unaffected seedlings to prevent smothering of the affected seedlings. In cases of treatment of growing tips very scrupulous selective pruning should be done to save affected sectors.
Q: What are some chromosome numbers in Rhododendrons?
The following information was published in the "Chromosome Atlas of Flowering Plants," by Darlington and Wylie, and is reproduced here by special permission: