2. Mutagenic action of nitrogen mustard.

Experiments on the mutagenic action of the nitrogen mustard, methyl‑bis (B‑chloroethyl) amine, on corn were begun in 1947 and continued in 1948. In general, the treatments consisted of exposing freshly collected pollen samples to vapors of the methyl‑bis (B‑chloroethyl) amine just before the pollen was applied to the pistillate parent. The exposure was made by introducing 0.2 ml. pollen samples, distributed uniformly over the flat bottom of a shallow 20 mm. diameter vial, into a chamber containing vapors of the nitrogen mustard.

TREATMENT - In 1947, the exposure was made in a desiccator containing a fan for circulating the air and a filter paper moistened with methyl‑bis (B‑chloroethyl) amine. For the 1948 experiments, the treating apparatus was redesigned to allow variation of both the vapor concentration and exposure time. This apparatus consisted of a 22 cm. U‑tube filled with very small glass helices covered with methyl‑bis (B‑chloroethyl) amine, a mercury‑filled gas burette, and a 300 ml. treating chamber. Dry air at the atmospheric temperature was drawn through the U‑tube where it became practically saturated with vapors of the B‑chloroethyl amine. Variable quantities of this dry air containing these vapors were then introduced through the gas burette into the treating chamber. This chamber contained a moist filter paper to maintain a high humidity and a magnetically operated fan placed so it would circulate the air‑vapor mixture through the pollen. The time of exposure of the pollen to the air-vapor mixtures was varied from one to five minutes, and the quantity of vapor introduced into the chamber ranged from one to 24 ml. The final concentration of methyl‑bis (B‑chloroethyl) amine in the treating chamber, as approximated from volatility data, probably varied from about one to 24 micrograms of amine per liter. Since the exposure time as well as the concentration was varied in the 1948 treatments, the "mortality product" or product of the concentration multiplied by the time varied from one to 120.

RESULTS ‑ Most of the seeds obtained in 1947 by pollinating normal plants with treated pollen were shrivelled. Many of these seeds, including some very small ones, proved to be viable and gave thrifty plants. The poor seed development was associated with grossly impaired endosperm formation. Tassel samples collected from the plants reared from these seeds were scored for defective pollen to obtain an estimate of the frequency of chromosomal abberations [sic] resulting from the treatments. One hundred eighteen plants among 760 sampled were scored as having a significant proportion of defective pollen. This is a frequency of 15.5 per cent. Among 58 controls none produced defective pollen. Seven hundred fifty‑six ears from self‑pollinated plants were harvested. Sixteen seeds from each were planted in the greenhouse and the seedlings seered for chlorophyll deficiencies. Five families segregated for such deficiencies. This is a frequency of 0.66 per cent. There were no chlorophyll deficient plants among the controls. A subsequent planting of 300 kernels per segregating family was made. The numbers of defective seedlings obtained from each of four families were 60, 68, 77 and 80. One ear had too few kernels for this test. The pollen treated in 1948 was from a a C R P Pl Pr B Y Su Sh Wx lg₁ stock. The pistillate parent to which the pollen was applied was a multiple recessive A C r^g p pl pr b y (Su su or su) sh vx Lg₁ stock. Use of pollen, which had been subjected to a treatment of a mortality product of one, resulted in almost normal appearing ears. Use of pollen, which had been subjected to a treatment of a mortality product of 120, resulted in partially filled ears with a high proportion of defective kernels. The kernels on these ears are now being classified for endosperm mutations. The following mutations in the endosperm have been observed: sugary, waxy, white, red aleurone and shrunken. Mosaic kernels are more frequent than mutations affecting the entire kernel. The frequency of mutations increases with the severity of the treatment. Plants will be grown from these seeds in 1949 and scored for mutations carried by the embryos.

Pryce B. Gibson, Lloyd Wilson, R. A. Brink, Mark A. Stahmann