12) If treated bud dies, watch for growth among lower laterals.
13) Evidence of changes appears in the new leaves, darker, thicker, greener.
14) Conclusive evidence of doubling rests with microscopic and anatomical analysis which is a task for trained technicians only.
The above procedures are suggestions for a start and everyone will wish to make changes suited to his particular needs. The concentration of colchicine need not be exact as in an analytical experiment in chemistry. One gram dissolved in 500 ml. water is an adequate and a sufficiently careful measurement. The local pharmacist or physician is well acquainted with colchicine in the practise of medicine since this drug is a standard for gout.
Effective use may be made from two specific areas of plant breeding. First, doubling of chromosomes changes sterile hybrids into fertile individuals. This is a promising field and whenever such hybrids are discovered, efforts should be made to apply the colchicine technique. Second, doubling of the chromosome number makes possible hybridization of individuals heretofore unsuccessful in such effort. In both instances germ plasm of wide genetic difference is incorporated into a new propagating breeding stock. In the case of the sterile hybrid transformed into fertile individuals, no counting of chromosomes is necessary because restoration of fertility is evidence of changes in the chromosomal makeup. However, the second type of experiment requires microscopic analysis.
There are a number of fundamental research problems in the plant sciences associated with the treatment of plants with colchicine. From horticultural subjects such as the apple,(3) pear, cranberries,(4) and grapes, it is obvious that periclinal chimeras will be of prime importance in analysis of results in treatment of nut trees. Following the treatment of a growing point with colchicine the outer layer of cells may be doubled by colchicine but the lower layers may remain unchanged. Or a reverse of this situation may obtain, and even other types. Since the formation of pollen takes place from a certain layer it is very important that such specific layers are changed. The course of plant breeding can be altered by these kinds of changes. To our knowledge, no investigations of periclinal chimeras have been made with nuts, following treatment with colchicine.
Specific experiments were conducted at Eldred, Illinois in the spring of 1951 with seedlings of pecan. The cooperation of the R.B. Best Farms and Nut Plantation made this project possible. Several types of treatment were tried. Out of this experience the above schedule listed in 14 steps was developed. Other details may be obtained by contacting the authors direct. Observations of the new growth in 1951 and 1952 were made and the shape of leaves, color, texture and general appearance suggest that doubling of chromosomes has been induced. Up until the present time, no microscopic analysis has been made but this is a contemplated step and facilities are at hand to complete this work.
While this paper is not a completed research, the authors hope that the presentation of technique will aid and stimulate interest in this new approach to nut improvement. In such instances where certain members may have a particular problem such as a true hybrid-sterile as a result of hybridity, it is hoped that the suggestions given in the above pages may lead into a new field of improvement. There are rewards in store for the plant breeder willing to master this new technique, but the mastery requires careful study and diligent work.
Literature Cited
1. Eigsti, O. J. and Dustin, P.—Colchicine Bibliography. Lloydia 10: 65-114. 1947.