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 rch 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 55C. 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 80C, 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 Pwr 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

 

X2

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 ‑ rg

x

fn ‑ rg

+  ‑rr

 

no recombination was obtained.

 

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

 

S. Horovitz

N. Horovitz