I'll just put this here as well...
Journal American Rhododendron Society
Current Editor:
Dr. Glen Jamieson
ars.editor@gmail.com
Volume 36, Number 3
Summer 1982
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TRIACONTANOL AS A GROWTH STIMULANT - A REPORT OF TWO EXPERIMENTS
By D.L Hinerman, M.D. and S.L. Kunkel, Ph.D., Ann Arbor, Michigan
Introduction
For many years beneficial effects of alfalfa on plants, animals and humans have been suspected. In the mid-seventies intense interest was generated by Stanley K. Ries et. al. when they observed significant increases in growth and productivity of plants which were in close proximity to alfalfa hay (Medicago sativa L). Further investigation led to the isolation of the active principle in alfalfa, 1-triacontanol. Alfalfa and triacontanol in small quantities not only stimulated growth and productivity of plants, but did so in the dark independently of photosynthesis.1,3,4,5,6,7,8
1-Triacontanol (CH3CH28CH2OH) is a ubiquitous long chain alcohol found incorporated in the surface of many plants and is a major component of beeswax.
Experiment #1 — Growth of Animal Cells
The effects of triacontanol on growth and productivity of plants is so striking that questions might be raised about similar effects on mammalian cells. With the great fears about environmental cancer-producing growth, it was considered wise to test triacontanol for growth promoting activity in mammalian cells.
Growing cells having active nuclear and cellular division with increase in DNA will incorporate thymidine, including radioactive 3H-thymidine. The process of nuclear and cellular division can be stimulated in cells by substances such as phytohemagglutinin (PHA). Cells treated with PHA then can be compared with similar cells treated with or without triacontanol. Quantitative counts of the amounts of radioactivity in each of the three groups of cells will reveal the relative amount of growth stimulation or DNA synthesis as shown by 3H-thymidine incorporation.2
This procedure was performed using cells derived from the spleens of CBA-J female mice from Jackson Laboratories in Bar Harbor, Maine. These spleens were removed by aseptic technique, were teased apart and gently pressed through a fine stainless steel mesh into a culture medium. The cells were 90-95% viable. Spleen cells were diluted to a concentration of 5x106/ml in culture medium with 10% fetal calf serum containing penicillin and streptomycin. To one series of cultures containing the above cells were added 0.001 to 10 µg/ml triacontanol solution, to another series 0.001 to 10 µg/ml of phytohemagglutinin, the known stimulant and to a third series nothing was added. All cultures were incubated for 60 hours, then 3H-thymidine was added to each of the cultures and incubated for another 12 hours.
Results
3H-thymidine incorporation
As shown in Figure I, there was no statistically significant increase in any of the triacontanol treated cultures above the non-treated cells. No increase was observed over a wide range of triacontanol concentrations 1 µg/ml to 0.001 µg/ml. On the other hand the PHA treated cultures responded in a normal fashion with a peak response at approximately 0.1 µg PHA/ml.
Fig. I. Incorporation of 3H-thymidine by mouse spleen cell cultures.
A) Triacontanol (Δ—Δ—Δ) over a broad concentration range
did not increase 3HdT incorporation over controls (●—●—●).
B) PHA (phytohemagglutinin) (o—o—o) demonstrated a
characteristic dose response curve with suppression of
3HdT incorporation occurring at high doses and a maximum
response at 0.1 µg PHA/ml.
Experiment #2 — Growth of Plants
Although some scientists and plant growers have seen remarkable increases in growth and production of plants from alfalfa and triacontanol, subsequent field tests by Ries and by other scientists from the U.S.D.A., universities and chemical companies have proved inconsistent and disappointing.5 Ries attributes most of the problems to the methods of formulation of triacontanol in that the long chain alcohol is very insoluble in water and in most other solvents. Ries and others have used combinations of solvents, detergents and surfactants in efforts to keep triacontanol in solution. The authors had a batch of triacontanol that had been opened for several months and this sample defied all attempts to get it in solution. A.J. Welebir is reported to have found additional requirements such as a solution at a pH of 8 or higher and the presence of metal ions such as calcium in the triacontanol solution. Using a solution of triacontanol with the above additions, Welebir has produced even more amazing growth and production than others have achieved and has done it consistently.5
Materials for experiment #2 consisted of eight first-year rooted cuttings (clones) of genetically identical plants of W. Delp's hybrid rhododendron (R. pink carolinianum x R. scintillans) #1. The cuttings were about the same size, consisting of one or two stems, small leaves and fairly uniform small root systems. They were placed in a rooting bed in a cold greenhouse (minimum temperature was 40°F.) The soil mixture was equal parts of peat and perlite. Bottom heat of 80°F was present. One mg of 1-triacontanol was dissolved in one ml of chloroform, carefully diluted with de-ionized water to make a 0.1 mg/liter solution. (Ries had found that aqueous solutions containing small amounts of chloroform had no effect on plants.) The pH of the solution was 5.3. The pH of the rooting mixture was 5.5. No calcium ions were present except in buffers found in the well-water (pH 7) with which the plants were sprayed at frequent intervals.
The trial began in mid-January when plants were dormant. Six of the eight plants were painted thoroughly with the triacontanol solution and this was repeated in one week and again in mid-April. In May the rooted cuttings were transplanted to individual pots. In June only four of the 8 plants were sprayed and in August only 2 of the 8 plants were sprayed making 5 the maximum number of doses for two of the plants.
Figure II illustrates the difference in growth between the total controls and the plants receiving 5 treatments. Five different measurements of growth were taken in early October.
Figure II Measurements of five different aspects of growth of rooted cuttings of a rhododendron hybrid, both untreated and treated with 0.1 mg triacontanol/liter.
TYPE OF MEASUREMENTCONTROL PLANTSPLANTS TREATED 5 TIMESINCREASE
Total Weight (minus soil)30.2 g83.1 g275%
Diameter of trunk at base4.0 mm8.0mm100%
Number of branches410150%
Number of leaves62167269%
Number of flower buds48100%
The first results of the experiment was an obvious premature break in dormancy within the first two weeks in the triacontanol treated plants (end of January), whereas dormancy did not end until early May in the control plants. After 2 months the growth in treated plants was double that of the untreated plants. At the end of the growing season the amount of growth roughly paralleled the number and duration of treatments. A fantastic amount of growth had occurred especially in those plants which had 4 and 5 treatments over most of the growing season. Those receiving 5 treatments had tertiary branching as compared to secondary branching in those with 3 and 4 treatments and primary branching in the remainder. The root system after 5 treatments appeared proportionately larger than other aspects of the plant. One of the most profound differences was that of a total weight of 2¾ times greater in maximally treated plants as compared to untreated plants. Of great interest also was the double number of blossom buds in maximally treated plants and the diameter of those buds was twice that of the untreated plants.
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