3. Phenol reaction on maize grains.

It is known that grains of many varieties of wheat are differentially colored after being soaked for a certain time in a weak phenol solution. The same reaction was tested on maize seeds of different lines, and it was found that some of them do not take color at all, while others develop a grade of color which is characteristic for each line. Results of reaction can be estimated in five grades, which we designate in increasing order of intensity from 0 to IV. Grade IV gives an almost black color like the one of r^ch gene producing "cherry pericarp".

The lines that do not develop color are very scarce. This reaction 0 is constant in the line and its progeny. In lines having pericarp colors it is difficult or even impossible to estimate the reaction. In the case of homozygous colored aleurone plants it is possible to obtain colorless aleurone seeds for the test, fertilizing the plant with pollen carrying I.

To make the reaction with maize, the procedure is as follows: In small flat‑bottomed test tubes, where a piece of filter paper is placed, one or two seeds from the same ear are introduced with two and one half per cent phenol solution, enough to cover the grains, allowing them to soak during 48 hours. After that period of time, the liquid is taken away and the tubes are covered with cotton wool until the grains acquire all the color they are able to develop. This happens after 48 hours at room temperature. After that, they are left to dry in the air on filter paper; then the observations are registered. As color develops, the pigment partially spreads from the grains to the surrounding liquid. If grains of grade 0 are present, they absorb color from the pigmented liquid. This is why grains from each ear must be tested separately.

Temperature has a marked influence on the speed of the reaction, the best being about 55�C. If treatment is made in ovens at this temperature, time for complete reaction is shortened considerably. In crosses of "phenol‑0" with "phenol color" and their reciprocals, the F₁ seeds give the reaction of the mother. All grains from the same ear give identical reaction, no matter which is the constitution of the mother plant or of the pollen used. It is, then, a pericarp character.

Reaction speeds up in the presence of an oxidant such as peroxide water. After two hours of immersion, the grains take a brown or chocolate color similar to the one that depends on gene Ch.

In grains heated before the test to 80�C, the ability to develop color is destroyed. It seems then that this character depends on the presence or absence of a diastase in the pericarp, which would produce coloration when phenol and oxygen are added.

Seeds retain for at least several years the ability to develop color. (We are indebted to Dr. R. A. Nico of the University of La Plata for the useful information on chemical aspects of this problem.)

Reaction was tested in other organs of the plant such as glumes, cob, pith, stalk, leaves and midribs, with negative results. In the growing seed, reaction is already apparent a week after fertilization and perhaps earlier. Grains occasionally formed in the tassel of phenol‑0 plants give a somewhat positive reaction.

We are transforming some ts stocks into phenol‑0, in order to study the environmental conditions responsible for that variation. The phenol‑0 stock used in crosses analyzed in this report came from a single sample of a genetic tester, a +/d, which was received from Dr. Brink, University of Wisconsin, in 1934, under the designation S‑367 sib.

In crosses of "phenol‑color" (I,II,III and IV) with "phenol‑0" the F₁ is colored, generally with an intermediate grade, but sometimes F₁ plants from the same cross reach grade IV, and other plants, grades I, II and III.

Number of cultures	Grade of parents crossed	F, plants
		Phenol reaction grades
		I	II	III	IV
1	IV x 0	2	1	4	6
1	0 x IV		1	2	6

The F₂ gives the ratio 3:1 between "colored" and "colorless" plants. In backcrosses to phenol‑0, these classes appear in a 1:1 ratio. Among the colored ones different shades appear. We suggest fn symbol (phenol‑0) for this character. The two following tables show the results of various crosses in F₂ and backcrosses. From the tables it can be inferred that the different grades are partly due to incomplete dominance and partly to modifiers of a principal gene (the dominant Fn) which conditions the presence of color. The hypothesis of multiple allelomorphs for colors is rejected.

Number of cultures	Grades of parents crossed including reciprocal	Grade of F1 plants	Phenol reaction in F2 plants						Totals
			Color grades				Total colored Fn	Colorless (Grade 0) fn
			IV	III	II	I
3	IV x 0	- *	90	26	5	3	124	35	159
5	III x 0	I	99	60	82	42	283	91	374
1	II x 0	I	1	17	24	33	75	32	107
1	I x 0	I	0	0	8	18	26	2	28
	Total (Fn/fn) selfed						508	160	668

* No grains were reserved for testing these particular plants.

Number of cultures	Type of backcross including reciprocal	Phenol reaction of F1 plants	Phenol reaction in progeny						Totals
			Color grades				Total colored Fn	Colorless (Grade 0) fn
			IV	III	II	I
3	(IV/0)/0	I; IV	35	2	1	0	38	39	77
5	(III/0)/0	I; II	5	19	14	28	66	63	129
1	(I/0)/0	I	0	0	0	4	4	2	6
	Total (Fn/fn)/fn						108	104	212

In the selfed progeny of crosses between different grades of "phenol color", as in crosses of F, by phenol‑0, all progenies are "colored". The two following tables show the data obtained in these crosses:

	Phenol reaction in the progeny
F1 Plant	Colored Fn	Colorless fn	Totals
(IV/II) selfed	102	0	102
(III/II) "	55	0	55
(III/III) "	42	0	42
Total (Fnx/Fny) selfed	199	0	199

	Phenol reaction in the progeny
Type of cross - including reciprocal	Colored Fn	Colorless fn	Totals
(IV/II)/0	245	0	245
(III/II)/0	54	0	54
(III/III)/0	111	0	111
Total (Fnx/Fny) /fn	410	0	410

In crosses with other genes, fn showed independence with P (allele P^wr was used), gl₂, B, a, d, Pl, gl, ij, sh.

Nevertheless, it seems linked with g of chromosome 10 in F₂ crosses -- in repulsion; summarized in the following table:

g +	selfed
+ fn

F2
+ + = 120	Segregation		X^2	P
+ fn = 53	G : g	:	0.428	0.70 ‑ 0.50
g + = 51	Fn : fn	:	0.120	0.80 ‑ 0.70
g fn = 1	f.linkage	:	16.043	very small

Recombination between G ‑ Fn = 14.5%.

As g is located at 14 map units from R in chromosome 10, its 14.5% recombination with fn in this limited experiment suggests the possibility that "phenolase in the pericarp" might be another pleiotropic effect of the member of the allelomorphic series of R. Adequate experiments to solve this point are under way.

The study is prosecuted for the identification of Fn modifiers, which alter coloration. One of these modifiers, which in certain crosses are responsible for the difference between grade 1 and deeper grades, is linked with aleurone color, perhaps due to C of chromosome 9.

Additional information (1947).

1. (Phenol reaction) fn is located to the side of r, and probably allelomorphic to it. In a limited population from the backcross:

fn ‑ r^g	x	fn ‑ r^g
+ ‑r^r

no recombination was obtained.

2. The phenol‑0 from Peru is allelomorphic with those previously found from other sources.

S. Horovitz

N. Horovitz