Some Notes on the Induction of Mutation in Rhododendrons
David Leach, Brookville, Pa.
The seeds referred to in these notes were treated as follows by Dr. Warren Spencer and Dr. E. G. Anderson (who was in charge of the radiation work on the Bikini atom bomb tests) at the Kerckhoff Laboratories of Biology at California Institute of Technology:
- Average target distance: 7.8 cm.
- 120 kilovolts, 8 milliamperes, unfiltered X-rays
- Total time of dosage: 6.71 minutes
- Average dosage received: 15,500 r. units.
As there are already many cells in the seed and a given mutation will be induced in only one cell, it is well to keep this in mind in attempting to recover any recessive mutations which might be induced. As this cell proliferates through division all of that portion of the plant derived from that cell can be expected to carry any mutation induced in the original cell. However, the other cells in the original seed could each carry an entirely different mutation so that other portions of the plant may have concealed mutations of widely varying character depending on the cells in the seed from which they ultimately formed.
The terminal buds of your plants should be removed after the plants are well started in order to get lateral buds which would have a much better chance of having homogeneous flowers as far as any induced mutant is concerned. If such flowers can be self-pollinated there would seem to be a good chance of getting the recessive mutation showing up in the second generation. All or several lateral buds should be tested as they all have an equal chance of carrying mutations. and different ones. I might suggest grafting as a means of speeding up the flowering of lateral buds.
Of course, if a dominant mutation is induced it will show up in some sector of the plant raised from your irradiated seeds. However, this is not probable. Such mutations as are induced will probably be of a recessive nature, and concealed; the recessive mutant will not appear until you fertilize the lateral blooms with pollen from the same flower and plants raised from such seed mature. This, of course, can not be done with species or hybrids which are self-incompatible. As I write the notes June 1, I have found with the dosage outlined above, there will be approximately 75% mortality of the seedlings and germination will be somewhat delayed.
Now regarding the colchicine, the procedure for treatment is as follows:
- Germinate the seeds on a moist, sterile paper towel in a glass-covered container.
- Transfer the seedlings just after they germinate into a shallow container, the bottom of which is lined with sterile paper towels kept thoroughly wet with a colchicine solution made by adding 1 gram of colchicine to 100 c. c. of water.
- Remove the seedlings at various intervals of time between 8 and 24 hours, rinse them well and transfer to flats for growing on.
Colchicine may be obtained from Benson-McLean Co., Bridgeton, Indiana, and some drug stores carry it. Do not breathe the solution, get it into your eyes, or on the skin. Use forceps.
The first effect of treatment is to dwarf the plant and malformed its leaves and branches, with some mortality to be expected. Here again, the effect of doubled chromosomes will not show up until the second generation. In this case, a microscopic examination of the flowers is the only sure way to determine in which flowers the chromosome number has been doubled. That is to say, the pollen of the flower should be examined by a suitable instrument. Those flowers in which the chromosome number has been doubled can be crossed and the effect of the colchicine treatment will then show up in seedlings raised from such a cross. This is the method which produced the famous tetraploid snapdragons, with much larger blossoms.
"Results of the treatment as described above will vary. In some cases a growth rate up to five times greater than normal will be observed, and in others the more typical malformations will appear."
The third method is the treatment of buds with colchicine. A solution of 0.4 of 1% of colchicine is made up in an emulsion of Hinds' Honey and Almond Cream diluted with an equal amount of water. The Hinds' Cream is merely a carrier. The growing buds are sprayed three times a week with the 0.4% colchicine in the thin emulsion. Treated buds are checked in growth and normal second branches are likely to grow out from neighboring untreated buds. Such second growth must be cut out or checked by further spraying with the colchicine emulsion. This treatment has resulted in double diploids in other genera and a similar effect might well be achieved with rhododendrons.
I believe the treatment of seeds with colchicine is preferable to spraying the buds. The treatment of seeds which normally produce sterile "mule" plants might be particularly rewarding if an adequate number of seeds were available with which to work.
Whichever method is used, if doubled chromosomes should be induced in two compatible fertile flowers the resulting seeds might produce startling results.
In either case, the colchicine is effective only while the cells are in the process of division in the normal process of growth. The colchicine prevents the migration to the new cell of the duplicate of the original chromosome which is formed as the cell prepares to divide; as a result the original cell, and all which subsequently form from it, have double the original number of chromosomes which it contained before treatment.
As you have probably observed from your own study of these matters, I have tried to simplify the techniques so that they could be used with equal effectiveness by the average rhododendron enthusiast who does not have a laboratory at his disposal. For example, the pricking out of the seedlings from the paper towel soaked with colchicine at intervals from 8 to 24 hours after placing them on the paper allows for a very wide latitude in tolerance among species and hybrids. There will probably be a higher mortality from the treatment but somewhere along the line in the time intervals the average amateur stands a good chance of obtaining the right treatment for the particular species or hybrids he is using.