Induction of mutations with nitrogen mustards.

Since 1946, a series of experiments were conducted to test the genetic effectiveness of tris - (B-chloroethyl) amine and di - (B-chloroethyl) methyl amine in maize. The methods of chemical treatment were: (1) soaking pre-germinated and dry seeds in 0.25% aqueous solution of the hydrochloride salt of the mustards at intervals of 2, 4, 6, and 8 hours; (2) exposing freshly shed pollen grains to the vapors of 0.4% solution of mustard salt buffered at pH-8.6, temperaturd 65� C, and (3) dipping the cut basal end of the tassel in a 0.25% solution of salts of both mustards at 10, 18, and 36-hour durations.

The usual technique for the identification of the genetic effects of mutagens was used, namely, a multiple dominant stock (C, Pr, Su, Lg₁, Bm₂ and B) was treated and the pollen grains applied to the silk of a multiple recessive stock (c, pr, su, lg₁, bm₂, and b). Endosperm deficiencies for dominant marker genes and dominant gene losses for plant characters are revealed by the appearance of the recessive characters in the F₁ seeds and F₁ plants respectively. The haplo-viability of the F₁ plants were checked by examination of the pollen grain fertility. Cytological examinations of sporocytes from plants exhibiting pollen sterility were made to determine the types of chromsomal aberrations induced by the mustards. Frequency of mutations for endosperm characters were determined from F₂ ears.

In the summer of 1948, X-ray and mustard gas treatments were carried out simultaneously so that the genetic effects of the two mutagens could be compared adequately.

Mutations were not obtained by seed treatment with the two mustards but there was an adverse effect on seed viability and phenocopies were produced. A high percentage of two-week-old seedlings arising from treated seeds exhibited a temporary chlorophyl-deficiency of the midrib. The 1208 plants that survived the seed treatment were all normal except for four plants that showed permanent sectorial chlorophyl deficiency which was not transmitted to the offspring.

The di - (B-chloroethyl) methyl amine proved to be more toxic to maize than the tris -(B-chloroethyl) amine. In most of the treatments where the former was used, the grain set was reduced to a minimum and seed abnormalities to a maximum. Although the mutagenic action of di -(B-chloroethyl) methyl amine is undoubtedly qualitatively the same as tris - (B-chloroethyl) amine, the fomer so reduced seed formation that the relation between dosage and genetic effectiveness was difficult to determine.

Tris - (B-chloroethyl) amine proved to be a very effective chemical mutagen when the multiple dominant tassel was dipped in a 0.25% aqueous solution of its salt. The results for the 18-hour treatment were as follows:

1947 - Seeds examined = 1963
Deficiencies for gene	C:	entire	- 9.4%;	sectorial	- 3.9%
	Pr:	"	-10.3%;	"	- 1.6%
	Su:	"	-14.8%;	"	- 2.5%
1948 - Seeds examined = 613
Deficiencies for gene	C:	entire	-16.0%;	sectorial	- 2.4%
	Pr:	"	- 5.2%;	"	- 0.7%
	Su:	"	-17.9%;	"	- 3.4%.

The 36-hour treatment of the tassel yielded the highest percentage of endosperm deficiencies, but due to the great reduction in set of grains (especially in the 1948 experiments), the results can hardly be considered significant. When the tassel was given a 48-hour treatment, pollen grains were not shed at all.

The three endosperm characters, C, Pr, and Su, showed equal vulnerability to tris - (B-chloroethyl) amine or conversely - the mustard action on these genes is not selective. When the sectorial deficiencies for the 10-hour, 18-hour and 36-hour treatments were considered, the results showed a definite trend for increase in the number of sectorials as duration of treatment was prolonged.

The endosperm character mutations were determined from ears in the F₂ generation following treatment with tris - (B-chloroethyl) amine. The average

mutation percentage for 10-hour, 18-hour and 36-hour treatments was for C - 1.6%, Pr - 3.3%, and Su - 1.4%. This mutation percentage for each gene should theoretically be equal to the percentage of the corresponding endosperm deficiencies because the male and endosperm gametes in the pollen grain should have the same chance of being acted upon by the mustard. But the endosperm mutations obtained were less frequent than the endosperm deficiencies observed. One of the feasible explanations for such a discrepancy is the low F₁ seeds viability which never exceeded 50% for any treatment. The mutation percentage shown by the F₂ seeds and F₁ plants is the minimum, for it represents only the mutations capable of survival in the environment in which these plants were grown. Many of the induced hereditary modifications may have been lethal but could not be detected. Attempts were made to grow the abnormal F₁ seeds in artificial media as excised embryos, and they were provided with the best envirorment our facilities could offer. Yet a high percentage of these seeds proved inviable. Of the 572 F₁ viable plants from the tris - (B-chloroothyl) amine treatments, 73 (12.7%) were semi-sterile. Cytological examinations of the sporocytes of these haplo-inviable individuals revealed the following chromosomal aberrations: translocations, inversions, deletions, laggards, sticky kinetochores and desynapsis of chromosomes at the first meiotic division. The cause of the defective pollen in other F₁ plants could not be detected cytologically. The number of semi-lethal plants and other morphological monstrosities is directly proportional to the duration of the treatment.

Forty-four F₁ progenies revealed dominant gene marker losses: Gene B - 3.8%; Lg₁ - 2.4%; Bm₂ - 3.1%. Fifteen of these 44 had normal pollen and it was considered that these losses could be gene or point mutations; whereas, the rest all exhibited haplo-inviability which can be due to deletions, asymetrical exchanges or non-union of breaks in the chromosomes.

The highest endosperm deficiencies induced by X-ray radiations of pollen grains were: Gene C - 6.3% from 2000 r; Gene Pr - 3.0% from 2250 r and Gene Su - 3.7% from 2000 r. This mutagenic capacity of the X-rays is roughly equivalent to the 10-hour dipping treatment of the cut tassel using tris - (B-chloroethyl) amine which effected the following deficiencies: C - 6.6%; Pr - 1.1%; Su - 5.8% The abundant production of sectorial deficiencies by mustard treatments, as contrasted to their scarcity in X-ray treatments, is a distinct difference between the genetic effects of the two mutagens.

Other chemicals were tested for mutagenic action. Acenaphthene, paradichlorobenzene, ethylene glycol, potassium thiocyanate and ethyl carbamate did not show significant endosperm deficiencies. But phenol, B-naphthyl amine, formalin, methylcolanthrene and ceepryn were considered to have mutagenic properties for maize, judging from the endosperm deficiencies they induced. Ceepryn proved to be very promising, since it produced an average of 9.8% endosperm deficiencies involving C, Pr and Su, computed on per-locus basis. From the results of the preliminary experiments on maize with these compounds the range of effective dosage has been determined.

D. L. Umali