Pollen which has been moistened in any way failed to function on the stigma. Consequently it was treated with the vapor of some chemicals which have an appreciably high pressure at room temperature.Formalin, ammonia, acetic acid, ethyl alcohol, amyl alcohol, ether and chloroform were selected to include protien coagulants, acids, bases and fat solvents. Some of these chemicals have been claimed to be mutagenic. Later, nitrogen mustard was included.
For the vapor treatment, the pollen were obtained on a clean dry slide. by shaking flowers with a buzzer, collecting from at least three to four flowers of different plants. The glass slide was then helt on an inclined plane, and lightly tapped to spread the pollen uniformly in a single layer. An aqueous solution of the chemical was used for the treatment. In the case of fat solvents like ether, chloroform and amyl alcohol, an emolsion was made by churning the mixture in an electric mixer immediatly before a treatment. The slide was kept above the level of the liquid by setting it on small glass rods. The inner rim of the petri-dish was smeared with vaseline to prevent diffusion of chemical vapor. The petri-dish was kept at 25 deg. C. five minutes before starting the treatment, so that the air inside would be saturated with chemical vapor. The slide was then placed inside the petri-dish by raising the upper lid from one side. Duration of exposure was varied, so that the chemical could penetrate the pollen to different degrees. Several concentrations of the chemical were tried. The effect of exposing the pollen grains to chemical vapor was observed by germinating them in acidified sugar, agar and gelatin medium (8 gms. of sucrose, 4 gms. agar and 4 gms. of gelatin in 100 cc. of water - Darlington and LeCour 1947). The medium was acidified with tartaric acid to 0.005 normal acidity, which was found to help germination of tomato pollen.
All the chemicals tested were found to inhibit pollen germination when treated in high dosages. The cause of such inhibition might be due either to formation of lethal genes or due to drastic changes in the chemical composition of the cytoplasm. A sublethal dose, where about ten percent of the treated pollen germinated, was found to vary within a narrow range for the different concentrations of a chemical. The exposure time for the sublethal dose of a chemical varied with the concentration.
Fruits were obtained when pollen treated at the sublethal dose was applied to emasculated flower buds. Pollen treated at doses that inhibited germination on the artificial medium, produced fruit in most cases. So the sublethal dose for fruit setting were determined directly by pollinating many flowers with the different concentrations of each chemical. This indicates that pollen which failed to germinate in artificial medium, did so on a more suitable medium of stigmatic fluid.
Methyl bis (B-chloroethyl) amine hydrochloride was used for the nitrogen mustard treatment. Ten milligrams of the compound was dissolved either in five or in ten millileters of distilled water and taken up in a hypodermic syringe. Sodium hydroxide was put in petri-dishes, one set having 5.5 cc. solution of 0.005 N., and the other set had 5.5 cc. of 0.01 N. alkali. The glass slide containing the pollen grains was placed within the petri-dish as described above and then 5 cc. of the solution was injected into the alkali from the hypodermic syringe. One of the set thus recieved 5 mgm. of the compound and the other 10 mgm., in 10.5 cc. of solution. The petri-dish was shaken for some time to increase reaction between the salt and the alkali, to form the active volatile amine. The pollen grains treated for a definite period were applied to emasculated buds of the tester stock. Enough pollen was applied to ensure fruit setting, since fruit setting was found to be very irregular even in lower doses when limited quantity of pollen was used.
Mutagenic effects of the agents were measured by determination of haplo-lethal segregations in the F1. Only nitrogen mustard was significantly effective in producing haplo-lethal F1 plants at the rate of 8 per 100. Of the other chemicals tested formalin and ammonia may prove to be mutagenic. Pollination with limited pollen seems to give an increase in mutation frequency. If the limited pollination technique is combined with study of populations larger than those used (2 to 400) it may be possible to obtain significant statistics for formalin and ammonia.