Hemerocallis J. 23(2):42–43. (1969)
AN "INJECTION" METHOD FOR TREATING HEMEROCALLIS SPIKES WITH COLCHICINE
W. QUINN BUCK, Arcadia, California
(Editor's Note: Colchicine, a drug obtained from a species of Colchicum, was originally used as a treatment for gout. It has since been found to inhibit or stop certain phases of cell division; while allowing the chromosomes to duplicate themselves, it keeps the cell itself from dividing into two new daughter cells. Thus the number of chromosomes are doubled within the cell. In the work described below, colchicine is applied to the cells which will form the male gametes and eggs of the flower; usually each of these cells has only one set of chromosomes (haploid), but, because of the colchicine treatment, they now have two sets of chromosomes (diploid). When these cells fuse during fertilization the resulting seeds will have four sets of chromosomes (tetraploid.). Typically tetraploidy results in plants that are larger, and often superior, horticulturally.—Gary Breckon).
During the years of experiment and trial which led to the writer's first colchicine-induced tetraploid Hemerocallis (daylily) hybrids in 1948, the use of the hypodermic syringe for treating the growing point was tried and abandoned as almost useless, because of the tremendous pressures needed to force even a small amount of the colchicine solution into the tissues. Dr. Traub's report3 of his success in polyploidizing daylilies by soaking small plants in colchicine solution demonstrated that the alkaloid was translocated within the plant, and this fact further spurred the writer's search for an "injection" method of treatment.
Study of Dermen's summary of colchicine techniques2, and personal observations in the writer's own work led to trials in the treatment of daylily flower spikes, with the hope of obtaining polyploid pollen for use on tetraploids already obtained. By 1955 these experiments showed that the diploid daylily flower spikes did offer a practical means of obtaining tetraploid pollen and seed after careful treatment by means of "injections" of colchicine solution given through glass tubes, which, in a fashion, served as hypodermic needles for transfusion of the colchicine. Latest trials, made with important refinements of the technique, showed it to be worthy of reporting to others interested in breeding tetraploid daylilies.
Very simply, this "injection" method consists of introducing the solution into the vascular system by means of glass tubes, as shown in our photograph. These tubes are made by rotating an eight‑inch piece of glass tubing in the flame of a fishtail Bunsen burner until the tubing fuses evenly and can be drawn to a fine point; two tubes of a convenient length are thus produced.
|Photo showing the method of attaching the glass tubing to the plant and the wire stake.|
Tubing of very soft glass, with an inside diameter of about six mm., is suggested. Small tubing is much easier to draw, but it can be extremely difficult to fill with the solution. The points should be just a bit larger than one mm. in diameter for best results. The points are very fragile, and many will be broken in use. Most of these points can be renewed by redrawing.
In attaching the tubes to the spikes, a slight puncture is made in the soft tissue of the young scape at a convenient position just below an immature bud; then a piece of tape is applied to the spike opposite the puncture. The tape must encircle the stalk and extend far enough up on the tube to hold it firmly in position, with the point in the puncture. (Masking. tape, three-quarters of an inch wide, or similar tape, can be used). The other end of the tube is taped to a wire stake for support; the stake must be stuck into the ground at the right spot, and at the right angle, to give this support without exerting a pull on the tube. The tube is then ready to be filled with aqueous colchicine solution, using an ear syringe.
The 0.2% colchicine solution very commonly used for treating plants is far too strong for our young spikes, and its use will result in high mortality of buds, or even of whole treated spikes. A concentration between 0.05% and 0.02% is suggested as a possible optimum for use in this "injection" method. The length of time that the tubes are kept attached to the spikes must be regulated by the amount of solution taken up; a period of twenty-four hours can be taken as a beginning, and the worker can shorten or lengthen this period as needed. Temperature and season are important factors to be considered here.
Tubes must be attached as soon as spikes are tall enough to make it possible, and before the buds have developed to a stage where it is no longer possible to get doubling of chromosome numbers in pollen and ovules. The point of the tube must be dipped into the colchicine solution just before insertion into the puncture and before the tape is applied to hold the tube in position; otherwise a small air bubble may block completely any passage of the solution into the spike.
Some skill in drawing out the glass tubes, a measure of manual dexterity in attaching the tubes and a great deal of trial and error under the worker's own conditions will be necessary. Possible results would make all this work worth while for those interested in serious work with tetraploid daylilies. Application of this technique to other herbaceous plants could be worked out readily.