NATIONAL INSTITUTE OF GENETICS(JAPAN)ANNUALREPORT

No.5(1954)

Published by

THE NATIONAL INSTITUTE OF GENETICS(Misima, Sizuoka-ken, Japan)

1955

Annual Reportof theNational Institute of Genetics
No.5,
'!."

1954

j

rt

Published by

The National Institute of Genetics (japan)
1955

.

,

CONTENTS
General statement . . .
Abstract of diary for 1954
Staff . . . . . . . .
Council . . . . . . .
Research program for 1954
Researches carried out in 1954
A. Human genetics
1. Genetics of microcephaly . . . . . . . . . . . . .
2. Partial sex-linkage in man. . . . . . . . . . . . .
a) Estimation of the frequency of partially sex-linked genesb) Unequal sex ratios among affected children of cousin
and non-consanguineous marriage in partial sex-linkage . . . . . . . . . . . . . . . . . . . .

1
4

5
6
7
11
11
11
11

12

B. Genetics and cytology cf some mammals.3. Further studies on T locus in the Japanese wild mom:e, Mus
musculus molossinus. . . . . . . . . . . . . . . 13
4. Test for allelism of alopecia periodica and furless in thehouse mouse (Mus musculus musculus). . . . . . . 16
5. H:stological and cytological studies of th~ testes of male
tortoiseshell cats . . . . . . . . . . . . . . . . 16

T

C. Cytology and genetics of tumors
6. A simple squash technique for observation of chromosome
structures in normal somatic and malignant tumor cells .
7. Chromosome constitutions in male germ cells of the rat .
8. Characteristics of V-shaped chromosomes occurring intumor cells of the YOSHIDA sarcoma . . . . . . . .
9. Nature of V-shaped chromosomes in tumor cells of MTKsarcomata II and III . . . . . . . . . . . . . .
10. Karyological characteristics of tumor cells of the Hirosaki
sarcoma . . . .
. . . ..
. ....
11. A further study on the transplantability of MY-momoe
sarcoma
12. Development of resistance to nitrogen mustard N-oxide in
the EHRLICH aEcites carcinoma . . . . . . . . . •

17
18
19
20
22
23
24

ii

13. Two spontaneous tumors occurring in inbred rats. . . . 25
D. Genetics of poultry
14. Sex-linked nervous disorder in the domestic fowl. . . . 26
15. Genetic variation and covariation in economic traits in some27
breeds of chickens .. . . . . . . . . . . . . .

E. Genetics and biochemistry of the silkworm and other insects
16. "Small egg," a mutant in the silkworm . . . . . .
17. Effect of temperature in the larval stage on the development of multilunar and multistar markings . . . . .
18. Genetical and biochemical studies on yellow lethal silkworm
larvae, III. On the property of phenol oxidase in thismutant. . . . . . . . . . . . . . . . . . . .
19. Comparative studies of yellow lethal lemon larvae (lem l )and albino lethal larvae (al) of the silkworm from theview-point of biochemical genetics . . . . . . . . .
20. Biochemical studies on the hibernating character in thephotoperiodism of a wild silkworm, Antheraea pernyi .
21. Biochemical and genetical studies on wild silkworm, II. On
the nature of the pigments in the epidermal tissues of
the Chinese tussar silkworm, Antheraea pernyi. . . .
22. Pterins found in the silkworm . . . . . . . . . . .
23. The relationship between eye pigment and pterins of Drosophila melanogaster . . . . . . . . . . . .
F. Population genetics of some insects and a land snail
24. Polymorphism found in some insects and in a land snaila) The lady-beetle Harmonia. . . . . . .
b) The lycaenid butterfly Neozephyrus taxilac) The land-snail Bradybaena . . . . . •25. Population genetics on the balanced polymorphism in Drosophila rufa. . . . . . . . . . . . . . . . . .
G. Population genetics of rice and barley
26. Analysis of genes for hybrid sterility in the varieties ofcultivated rice . . . . . . . . . . . . . . . . .
27. Variation in fertility due to gametic-development genes
28. Change of segregation ratio due to gametic-development
genes. . . . ... . . . . . _ . . . . . . . .
29. Change of gene frequency in hybrid populations of rice

28
29

30

32
33

34
35
36

37
38
39
40

40
42
44
45

iii

30. Restriction of gene recombination in hybrid populations ofrice . . . . . . . . . . . . . . . . . . . . .
31. Do chromosomes in tetraploid hybrids between distantly
related varieties of cultivated rice tend to pair selectively?
32. Genetic analysis of differentiation of local strains in thebarley " Hosogara No.2"a) Statistical differences among local strainsb) Difference in growth habits among local strainsc) Competitive ability of local strains . . . . .
H. Studies on competition
33. Effect of the number of competing and non-competing individuals on competitional increment . . . . . . . .
34. Polygenic analysis of quantitative characters under the influence of intergenotypic competition. . . . . . . .
35. How does a mixed population of autogamous plants change
in response to intra-population competition?
. . . .
36. Competition experiment with diploid and autotetraploidraces of rice . . . . . . . . . . . .
37. Competition experiment with two Nz"cotz"ana species andtheir allotetraploid... . . . . .

47
48

50
51
52

53
54
56
58
59

1. Radiation genetics of wheat
38. Gene mutations in Einkorn wheat induced by X~rays . . 60
39. Chlorina mutants in Einkorn wheat induced by X-irradiation . . . . . . . . . . . . . . . . . . . . . 61

J. Cytology and genetics of Nicotz"ana
40. Cytogenetic studies on the genus Nzcotz"ana, VI.
a) Reduction divisions in hybrids between N. tabacum andtwo other species. . . . . . . . . .
b) Reduction divisions in several hybrids between Nz"cotz"ana species . . . . . . . . . . .
c) Reduction divisions in N. LangsdorjJiz". .
41. Mutation in tobacco plants induced by X·rays

62
64
65
66

K. Genetics, cytology and biochemistry of some phanaerogams
42. Hybridization experiments with Citrullus vulgaris andC. colocynthis . . . . . . . . .
43. Karyotaxonomic studies in Poaceae, II . . . . • .

67
68

iv

44. Effects of extracts from two poisonous plants upon living
plant tisst:ea) Extract from Gloriosa superba . . . . . . . . . .
b) Extract from Zephyranthes candida. . . . . . . .
45. Analysis of flavone pigments in Triticum and related plants
under consideration of their genome constitution . . .
46. Pa~er-chromatographic analysis of anthocyanins occurringin several varieties of Japanese morning glory with specialreference to the role of mg allele . . . . . . . . .
47. Studies on natural anthocyaninsa) Red coloring matter in the leaves of Perilla varieties.
b) Anthocyanin of purple·red flowers of Lespedeza. . .
c) Survey of anthocyanins responsible for the autumnal
reddening of leaves in Japan . . . . . . . . . .
L. Cytology and genetics of some lower organisms
48. The delayed appearance of the O-antigen transformationin Salmonella . . . . . . . . . . . . . . . . .
49. The paths of the reversion in the methionine-requiring
strain of Ustz'lago maydi's . . . . . . . . . . . .
50. Cytoplasmic polyhedral virus occurring in the silk",orm .
51. Studies on the mitochondrial granules isolated from Paramecium caudatum . . . . . . . . . . . . . . .
52. Fine structure of mitochondria in Paramecium. .'. . .
53. Studies on the lysogenicity of Pseudomonas solanacearum.
a) Lysogenic strain T·c 200 . . . . . . . . . . . .
b) Response types shown by the T-13 bacteria after exposure to S-9 phage. . . . . . . . . . . . . .
c) A double lysogenic strain . . . . . . . . . . . .
54. Studies on the multiplication of bacterial virus affecting
Streptomyces griseus . . . . . . . . . . . . . .
55. Electron-microscopical studies of ultra·thin sections in Pent"..
dllium chrysogenum. . . . . . . . . . .

69
70
72

72
74
74
75

75
77
78
79
79
79
80
80
82
82
83

M. Theoretical studies on breeding
56. Some considerations on the problem of secondary selection
in self·fertilized crop plants. . . . . . . . . . . . 84
57. Theoretical basis for discriminating the most desirable hy·
brid combinations in early generations . .
86

Books and papers published in 1954 by staff members

88..

The Emperor examining a microscopic preparation

GENERAL STATEMENT•

1954 was an eventful year for the Institute. Fortunately, all themain events were welcome ones. A new Department, that of Applied
Genetics, was added to the preexisting four departments. This new
department has two Laboratories, one of Applied Animal Genetics
and the other of Applied Plant Genetics.
On June 1, the Institute celebrated its Fifth Anniversary, withthe attendance of more than two hundred guests and about eighty
members of the Institute. The Director made a report on the pasthistory and present status of the Institute. Several guests spoke
congratulatory remarks. After the ceremony, the guests were shown
the buildings, equipment and experimental fields. Public lectures by
the staff were given in Tokyo and Numazu in commemoration of thiscelebration.
On October 18, the Institute received a group of 48 visitors who
were participants in the Congress of the Food and Agricultural
Organization of the United Nations which was in session in Tokyo.
Next, on November 4, the Institute was honored by the visit of
H. 1. M. the Emperor. The Biologist Emperor spent five busy hours

2

GENERAL STATEMENTin inspecting equipment and hearing reports on researches from thestaff. He showed keen interest in these talks and demonstrations.
Hiko-Ichi OKA, D. Ag., Yukio YAMADA and Takatada KAWAHARA
were newly appointed researchers during the current year, OKA as alaboratory head in the Department of Physiological Genetics, while
YAMADA and KAWAHARA belong to the new Department of Applied
Genetics. Katumi TANAKA, D. M. was appointed Research Associate;
his specialty is human genetics.
SAKAI was promoted to the Head of the Department of Applied
Genetics. MATSUMURA was sent abroad by the Government. He
spent seven and a half months in the United States and Europe,
visiting various genetics institutes and inspecting equipment andresearch projects, primarily those concerned with radiation genetics
and polyploidy breeding. He also attended the Seventh BiologicalResearch Conference held in April, 1954 at Brookhaven National
Laboratory N. Y., U. S. A. and the Eighth International BotanicalCongress held in July, 1954 at Paris. YOSIDA was awarded D. Sc.
from the Hokkaido University. KIMURA took a leave of absence foranother year to study mathematical genetics in the Department of
Genetics of the University of Wisconsin. IINO also received a leaveof absence for a year to study biochemical genetics in the samedepartment of the University of Wisconsin.
The whole set of buildings and equipment for poultry breeding, as
well as a residential house, which had belonged to the Whole-Japan
Association for Poultry Genetics, were donated by the Association to
the Institute for the use of the Department of Applied Genetics. The
building for silkworm culture was extended to 81.71 tubo. The newsection, 50 tubo in area, has some new arrangements which are rarely
found in other sericultural houses in the country. A 30 tubo housefor the temporary storage of crops from the experimental field wasconstructed. Two new residential houses were added to the group ofsuch houses located in the detached campus.
The library has been steadily expanded by the acquirement of
new books, periodicals and reprints. Many of these were given byscientific institutes or associations, or by individuals. Especially, Dr.
GOLDSCHMIDT has continued to send in reprints and current numbersof various scientific journals which amounted to 1,079.
The mousery has acquired 15 new strains of mice from the.'GE:'ERAL STATEMENT

3mouseries of Oxford University, Ohio State University and the Natio~
nal Institute of Preventive Hygiene in Tokyo. Various new strains ofwheat, Aegilops, sugar beet, morning glory, Ids and radish wereadded to our collections.
Among the foreign visitors, besides those in the group of F AO
congress participants, were: Dr. R. R. GATES of Harvard University;
Mr. Khin MA'0:-lG and Mr. San PE, Burmese rice breeders; Dr. R. A.
SILOwand Dr. K. RAMIAH of FAO; Dr. L. N. H. LARTER, British plant
breeder stationed at Singapore; Dr. N. PARTHASARATHY of the Central
Rice Research Institute of India; and Dr. D. P. RAICHOUDHURY, Mr. C. L.
VERNA and Mr. S. C. DAS, Indian silkworm geneticists and breeders.
The following grants were received during the current year by
our staff. These grants have been of great aid to our research
projects.
From the Fund for Grants-in-Aid of the Ministry of Education t:>
Cooperative Investigations: to K. OG~MA and co-workers (including
T. KaMAl, S. MAKINO, T. H. YOSlDA and K. TUTIKAWA), for: Cytologicaland genetical researches on tumors- ¥ 670,000; S, MATSUMURA andco-workers, for: Researches on the physiology of crop plants understandardize:! temperature, humidity and day-light conditions-¥ 430,000:
Y. TAKENAKA and co-workers, for: Genetics of morning glory¥250,OOO.
From the Fund for Grants-in-Aid to Investigations in Applied
Sciences: to Y. TANAKA, for: Research on artificial control of diapause
in the wild silkworm, Antheraea pernyi- ¥ 360,000 ; M. TsuJITA, for:Researches on the effect of environment in the manifestation ofhereditary characters in the silkworm and its practical application tobreeding-¥ 90,000.
From the Fun:! for Grants-in-Aid to Individual Workers: to M.
TSUJITA, for: Ele:::tron-microscopical studies on the cytology of thesilkworm- ¥ 80,000; M. TsuJITA, for: Cyto-genetic studies on micro·organisms by me:lns of ultra-thin sections- ¥ 60,000; K. SAKAI, for:Population-genetic studies on the contamination of rice crop by
mixture of "red rice"- ¥ 90,000.
From the Fund for Grants-in-Aid to Young Research Workers, to
K. GOTJH, for: Genetics on the mechanism of racial differentiation of
the strain "Hosogara No 2" of barley - ¥ 27,000; T. ENDO, for:
Bio::hemical genetics of flower colors of Viola tricolor-¥ 25,000;

4ABSTRACT OF DIARY FOR 1954

S. TSUDA, for: Studies on the propagation of bacteriophages- Y 25,000 ;B. SAKAGUCHI, for: biochemical-genetical study on pterin metabolism insilkworm - ¥ 27,000; S. NAWA, for: Chemistry of pterin occurring in
insects and its genetics-¥ 20,000; T. ISHIHARA, for: Cytological andhistological studies on the testis of male tortoiseshell cat-¥ 15,000;
C. MATSUI, for: Genetic study of bacteriophages-¥ 20,000.
From the Fund for Grants-in-Aid for Promotion of Improvementof Agricultural Techniques of the Ministry of Agriculture andForestry: to K. SAKAI and co-workers, for: Comparative studies
between pedigree method and bulk method in plant breeding¥ 95,000.
(Taku KaMAl)

ABSTRACT OF DIARY FOR 1954
Jan. 14. Tenth meeting of the Board of Cour:ciIIors.
Jan. 22. Twenty-third meeting of Misirr.a Geneticists' Club.
Feb. 13. Board meeting of Association for the Propagation of theKnowledge of Genetics.
Feb. 22. Twenty-fourth meeting of Misima Geneticists' Club.
March 25. Twenty-fifth meeting of Misima Geneticists' Club.
April 23. Twenty-sixth meeting of Misima Geneticists' Club.
May 17. Symposium on Population Genetics.
May 24. Twenty-seventh meeting of Misima Geneticists' Club.
June 1. Ceremony of the Fifth Anniversary of the Institute.
June 9. General meeting of Whole Japan Association of Poultry
Genetics.
June 22. Eleventh meeting of the Board of Councillors.
June 25. Twenty-eighth meeting of Misima Geneticists' Club.
Aug. 7. Public lectures in commemoration of the Fifth Anniversary in
Numazu City Hall.
Sept. 20. Twenty-ninth meeting of Misima Geneticists' Club.
Oct. 8. Thirtieth meeting of Misima Geneticists' Club.
Nov. 4. H. I. M. the Emperor's visit.
Nov. 5. Public lectures in commemoration of the Fifth Anniversary inY omiuri Hall in Tokyo.
Nov. 13. Meeting of Arrangerr.ents Committee for the International
Genetics Symposia.
Nov. 19. Thirty-first meeting of Misima Geneticists' Club.
Dec. 20. Thirty-second meeting of Misima Geneticists' Club.

STAFF

5

STAFF
Department and Laboratory Heads
Kan OGUMA, D. Ag., Director
Yoshimaro TANAKA, D. Ag., D. Sc., Head of Department of Morphological Genetics
Taku KOMAI, D. Sc., Head of Department of Physiological GeLetics
YO T AKENAKA, D. Sc., Head of Department of Cytological Genetics
Mitsuo TSUJITA, D. Ag., Head of Department of Biochemical Genetics
Kan-Ichi SAKAI, D. Ag., Head of Department of Applied Genetics
Seiji MATSUMURA, D. Ag.
Tosihide H. YOSIDA, D. Sc.
Kozo HAYASHI, D. Sc.
Hiko-Ichi OKA, D. Ag.

Part-time Staff and Research Associates
Hitoshi KIHARA, D. Sc., Professor of Kyoto University
Sajiro MAKINO, D. Sc., Professor of Hokkaido Cniversity
Yosito SINOTO, D. Sc., Professor of International Christian University
Hideo ETO, D. M., Assistant Professor of Tokyo University
Kazuo FURUSATO
Yoshinari KUWADA, D. Sc., Emeritus Professor of Kyoto University
Flora Alice LILIENFELD, Ph. D.
Yasunosuke OZAKI, D. M.
Katumi TANAKA, D. M.

Junior Investi"gators
Moto KIMURA, on leave of ab:oence
Kanji GOTOH
Toru ENDO
Akira MIYAZAWA
Kiyosi TUTIKAWA
Bungo SAKAGUCHI
Tetuo IINo, on leave of absence
Takatada KAWAHARA
T~shifumi TAIRA
Saburo NAWA
Takaaki ISHIHARA
Seizo TSUDA
Yukihide ABE
Sadao SAKAMOTO
Tsuguo T ATEOKA
Kimiji ONIMARU
Yukio YAMADA

Assistants-5
Department of Administration
Kan·ichi OTOFUJI, Head of Department

COUNCIL

6

Sumiyoshi SUGIO, Head of General Business Section
Masao MIYAZAWA, Head of Finance Section
Hiroko NAKANO
Junzo KADOWAKI
Naomi MATSUBARA
Clerks, TYPist, Telephone operator, Chauffeur, Ft"eld laborers, jant"tors, etc.-I8

Mz"st"ma Branch of Hatano Tabacco Expert"ment Statzon
Masao TANAKA, Head
Flora Alice LILlE~FELD

Seiji IMAI
Assistants-4

Whole-japan Association of Poultry Genetics
Kan OGUMA, President
Yoshimaro TANAKA, Vice President and Director of Researches

Associatzon for P,'opagation of the Knowledge of Genett"cs
Kan OGUMA, President
Yo T AKEi-<AKA, Managing Director
Seiji MATSUMURA, Managing Director

COUNCIL

Yo K. OKADA, Director of National Science Museum, Chairman
Seishi KAYA, Professor of Tokyo University, Vice Chairman
Tanemoto FURUHATA, Professor of Tokyo Medical and Dental College
Eikichi HIRATSUKA, Member of Japan Academy
Takeo IRUMANO, President of Japan Monopoly Corporation
Seiw KATSUNVMA, President of Nagoya University
Riichi KAWAKAMI, Head of Department of Biometry, National Instituteof Public Health
Kiyoshi MASUI, Professor of Nagoya University
Toshitaro MORINAGA, Director of Agricultural Technique Institute
Masanori NAKAIZUMI, Professor of Tokyo University
Toshio SAITO, Governor of Shizuoka Prefecture
Kin-ichiro SAKAGUCHI, Professor of Tokyo University
Takeo SAKATA, President of T. Sakata & Company, Yokohama
Mas3.tada TERADA, President of Jikei Medical College
Bungo WADA, Professor of Tokyo University

RESEARCH PROGRAM FOR 1954

7

RESEARCH PROGRAM FOR 1954
DEPARTMENT OF MORPHOLOGICAL GENETICSFirst Laboratory (TANAKA)
Stu::lies on unstable genes in the silkworm (TANAKA)
tests in Bombyx mort~ for making up chromosome maps
(TANAKA)
Experiments on the control of diapause in Antheraea pernyi (TANAKA)
Genetical studies on the Japanese long-tailed fowl (TANAKA)
Linkag~Second Laboratory (MATSCJMURA)
Radiation genetics in whe3.t and barley (MATSCJMURA and FUJII)
Studies on Agropyron, a genus related to Triticum (MATSUMCJRA andSAKAM::JTO)
Nullisomic dwarfs in the offspring of pentaploid wheat hybrids(MATSCJMCJRA)
Mutations in tobacco induced by X-ray (MATSUMURA, KIHARA and
FUJII)
Relation between the induced mutation and the quality of radiation(MATSUMCJRA ond ETO)
Physiological anj genetical studies of crops under control of temperature, moisture and day length (MATSUMURA et al.)Improvement of crops by means of induced triploidy (MATSUMURA et al.)Third Laboratory (KaMAl)
Genetics of human microcephaly (KaMAl, KISHIMOTO (Nagoya Univ.)
and OZAKI)
Partial sex-linkage in man (K. TANAKA)
DEPARTMENT OF CYTOLOGICAL GENETICSFirst Laboratory (YosIDA)
Cytological study of tumors (YosIDA and ISHIHARA)
Study on differentiation of sex-chromosomes CYosIDA)
Genetical study on the tumor sus~eptibility of animals (YosIDA and
ISHIHARA)
Study on development of resistance of tumor cells to anti-cancer agentsCYosIDA and ISHIHARA)

8

RESEARCH PROGRAM FOR 1954

Histological and cytological study of the testes of male tortoiseshellcats (ISHIHARA and YOSIDA)

Second Laboratory (T AKZ~AKA)
Origin of sex differentiation in higher plants (TAKENAKA)
Induction of abnormal mitosis and inhibition of growth by substances
extracted from certain plants (TAKENAKA)
Cytogenetics of Nicotiana (TAKENAKA, FURUSATO and LILIENFELD)
Cytogenetics of Citrus (FURUSATO)
Genetic studies of Pharbitis Nil (TAKENAKA)
Collection and preservation of useful strains of plants (TAKENAKA)
Classification of Gramineae by karyotypes (TATEOKA)

Third Laboratory (TSUJITA)Electron-microscopic studies on minute structures of cells of the silkworm (TSUJITA and TSUDA)
Electron-microscopic studies of microorganisms (TSUJITA and TSUDA)
DEPARTME~T

OF PHYSIOLOGICAL GE~ETICS (KaMAl)
First Laboratory (KaMAl)
Population genetics of the lady-beetle, Harmonia axyridis (KaMAl)
Population genetics of the butterfly, Neozephyrus taxila (KaMAl)
Population genetics of the land-snail, Bradybaena simz1aris (KaMAl)
Population genetics of Drosophila rufa (TAIRA)The T locus in Mus musculus molossinus (TUTIKAWA)"Alopecia pedodica" in mice (TUTIKA\VA)
Tumor susceptibility in mice (TUTIKAWA)
Breeding and preservation of mouse and rat strains useful for medical
investigations (OGUMA, MAKINO and TUTIKAWA)

Second Laboratory (OKA) and Third Laboratory (SAKAI)
Analysis of genes respcnsible for hybrid sterility in rice plants (OKA)
Analysis of genes controlling temperature respose in germination ofrice plants (OKA)
Heritability of various characters and genetic correlaticns in rice plants(SAKAI and OKA)
Population-genetical studies on wild rice plants (OKA)
Physiological-genetical studies on cultivated plants under regulated

RESEARCH PROGRAM FOR 1954

9temperature, humidity and day-light conditions (MATSUMURA et al.)Local differentiation of races in barley (GOTOH)
DEPARTMENT OF BIOCHEMICAL GENETICS (TS0JITA)First Laboratory (TSUJITA)
Biochemical-genetical studies on pteridine compounds in insects(NAWA)
Embryological and genetical studies of the silkworm (TSUJITA andSAKAGUCHI)
Genetical and biochemical studies of some insects (TSUJITA andSAKAGUCHI)

Second Laboratary (HAYASHI)
Chemical analysis of coloring substances of various varieties of morning glory and their genetic behavior (HAYASHI and ABE)
Chemical composition of pigments in autumn leaves and alpine plants(HAYASHI and ABE)
Biochemical-genetical study on the colors of Viola tricolor (ENDO)
Bicchemical studies of color variation in flowers (HAYASHI)
Relation between flavonoid pigrr.ents and the corresponding genome in
Triticum and allied species (ENDO)

Third Laboratory (HAYASHI)
Biochemical genetics of Ustz1ago (lINo)
Transduction and transformation of antigen type in Salmonella (lINO)
DEPARTMENT OF ApPLIED GEKETICS (SAKAI)First Laboratory (TANAKA)
Breeding for high egg production in poultry (TANAKA and KAWAHARA)
Genetics of hereditary tremor found in Plymouth-Rock fowl (KAWAHARA)
Heritability and genetic correlation of economic characters in poultry(YAMADA)On color changes of Plymouth-Rock fowl (TANAKA)
Variation in quantative characters in inbred animals (YAMADA)

Second Laboratory (SAKAI)Studies on competition between individual plants of different genetic
composition (SAKAI et al.)10

RE3EARCH PROGRAM FOR 1954

Theoretical and experimental studies on selection in autogamous plants(SAKAI)
Genetics of "red leaves" in the tobacco plant (SAKAI and IYAMA)
Genetics of quantitative characters in eggplant (GoTOH)
On the hybrid between water-melon and Citrullus colocynthis (FURUSATO and MIYAZAWA)

Research Students and Research Subjects
Mizuho YOSHIDA: Genetics of silkworm
Setsuji KATAOKA: Cytogenetics of plants
Seiji TEWKA: Cytogenetic studies on tobacco plants
Kozo NAKAMURA: Cytogenetics of Agropyrum
Yasuo OrA: Cytogenetic studies on morphological characters of Citrus
Taro AKIYAMA: Genetics and breeding of Citrus
T6y6 MITARAI: Fundamental morphological studies of animal cells
Toshio OMURA: Cytology of cancers
Kyoz:J WATANABE: Cytogenetical studies on Paramecium
Chiaki MA'SUI: Morphological studies on transformation of bacteriophages
Osamu YOSHIZAWA: Genetical studies on bacterial viruses
Shin-ya IYAMA: Population-genetics of upland rice
Yasuo SUZUKI: Population-genetics of crop plants

RESEARCHES CARRIED OUT IN 1954
A. HUMAN GENETICS

1. Genetics of Mt'crocephaty(by Taku KoMAI)

The study which bad been in process for some years in cooperation with K. KISHIMOTO, M. D. of Nagoya University and Y. OZAKI,
M. D., Research Assoc:ate of our Institute, was completed and sent
to press during the current year. The material consisted of 93 malepatients and 50 female patients. This was analyzed by using
HALDANE'S method (1938). Our original view that the great majorityof these cases are probably due to a recessive autosomal gene has
been sustained. The dominance of the normal gene over the genefor microcephaly is nearly always complete, but in exceptional cases
the gene for abnormality manifests itself to a slight extent. The
possible cause of the striking preponderance of males among thepatients was looked for by making a distinction between the" primary"cases (probands) and" secondary" cases in the sibship. Thus, it has
been disclosed that this sexual disparity among the patients is
probably due in a large measure to a "social" cause in that malepatients are more readily found outside their homes than female
patients and become the "primary" cases.
Neither the birth order nor the mother's age seems to exert any
influence on the production of a microcephalic child.
As previously reported, the incidence of the gene for microcephaly
among the Japanese people is 0.0034-0.0063, and the rate of new
mutation of this gene has been estimated as (2.20-7.57) x 10- 5•
Reference: KOMAI, T., KISHIMOTO, K. and OZAKI, Y. 1955. Genetic studyof microcephaly based on Japanese material. Am. Jour. Human Genet. 7: 51-65.

2. Pal'tiat Sex-linkage in Man(by Katumi TANAKA)a) Estimation of the frequency of partially sex·linked
genes. The frequency of an autosomal recessive gene may be givenby the formula:

12

RE3EARCHE3 CARRIED OUT IN 1954

r=c(1-k)/(16k-15c-ck)(1),where k and c are the rates of first cousin rr.atings among the parentsof the affected individuals and in the general population respectively.
This formula is based upon the expectation that the probability ofobtaining a homozygous child from a first-cousin mating is given by(r/16 + 15r2j16). Formula (1) is not applicable to partiaIIy sex-linked
genes, since the probability is variable according to crossover values
and the type of cousin mating.
The probability of getting an affected child in the mating inwhich the couple are the children of brothers (type 1), when acommon grandparent of the cousins had the gene in question, is given
by d 1=pq(1+2p2+2q2)/8, that in the mating of the children of sisters(type 2) by d z=(l+2p2+2q2)/32, that in the mating of a son of abrother and a daughter of a sister ~type 3) by d 3=PCl+4q2)/16, andthat in the mating of a son of a sister and a daughter of a brother(type 4) by d 4=q(l+4p2)j16; where p denotes the crossover value ofthe gene and q=l-p.
The frequency of a partiaIIy sex-linked recessive gene may be
estimated more accurately by means of the folIo wing formula:4(l-k) ~ diCi

r=

i~I

4
(l-k) ~ diCi+k-c.................. (2),i~lwhere Cr. Cz, C3 and C4 are the incidences of the four types of cousin
marriages respectively in the general population. Actual instancesof 466 cousin marriages coIIected from various parts of Japan are
distributed in the proportions cl=18.9%, cz=30.0%, c3=23.2% and C4=
27.9%.
When these relations are taken into consideration, values 12-15%
lower than those in NEEL et a!.'s estimations are obtained as thefrequency of the gene responsible for total color-blindness.
b) Unequal sex ratios among affected children of
cousin and non-consanguineous marriages in partial sexlinkage. It is expected that for partialIy sex-linked traits there
should be an excess of affected daughters over affected sons among
the children of consanguineous marriages. However, no indication ofsuch a tendency seems to have been found in the actual data.
The author has coIIected cases of congenital total color-blindness

RE>EARCHES CARRIED OUT IN 1954

13

from the Japanese literature, and also through personal communications with clinicians. The data are summarized in the table.
Relationship of parents

Number of affected children
Total

(a)
(b)

First cousins
Non-consanguineous

76
38

Males
36
31

Females Sex-ratio
40
0.90
7
4.45

The sex ratio among affected children of cousin matings (group a)
indicates only a slight excess of females, the difference being notsignificant. However, there is a remarkable excess of·males among
affected children of non-consanguineous matings (group b), the difference between groups (a) and Cb) being significant at the 0.1% level.
It appears thus that consanguineous matings produce relatively more
affected females than non-consanguineous matings. More carefulanalysis has revealed that the excess of male patients among theoffspring of non-consanguineous matings is only due to the tendency
that more male patients than female patients are ready to visit
hospitals in large cities, from which the great majority of the data
are derived.

B. GENETICS AND CYTOLOGY OF SOME MAMMALS

3. Further Studies on T Locus in the Japanese Wild Mouse, Mus
musculus molossinus(by Kiyo3i TUTIKA w A)

To examine whether the locus T in the Japanese wild mouse, Mus
musculus molossinus, is also mutable as in the other subspecies, Musm. musculus, wild mice caught in three localities were tested for thealleles at this locus.
The method of identifying t-alleles in molossinus populations waspreviously reported (Ann. Rep., No.4: 13, 1953). Small samples ofwild mice obtained from Misima, Niigata and Fukuoka were tested.
All these samples contained some animals heterozygous for at-typeallele similar to that in musculus.
Wild mice from Misima-The samples were from two sources:a male (9515) and a female (9613) caught in a rice-field at Kawa·
haragaya, Misima, and several males and females trapped in thebarn in our Institute. Of the eighteen males and nine females tested,

14

RESEARCHES CARRIED OUT IN 1954

Table 1. Results of testing mice of wild population Misima.
----

Wild
parentF)
NormalBrachy

10

13

4
2
2
2

4

Tailless(;WildparentF)
NormalBrachyTaillessI(

m-129
m-1331m-30l2m-3611m-410
m-42131m-42133
m-42134
m-4227m-425
m-43121m-4321
m-444
m-4462m-8529

9
3
5
5
7
12
3
6
19
4

Totals from15 (; (; +/ +

93

m306
m-802
m-951510
3

1

5

Totals from
3 (; 0 +t m ?

17

1

25

1
3
3
7
2
3
4
2

2

9
4
6
7
4

21

72

23

4

m-185
m-21029
m-318
m-42132
m-43122m-4463m-5323m-9613102
1
2
1

Totals from
8 I( I( +/+~843m-4411

7

1

1

Total from19 +/tm

7

1

1

8

5

9
5

11

10
6
3
2
6

6
14

five males and one female produced some tailless offspring (Table 1).
Five of these tailless mice produced by a male (4462) was tested
against the tailless line (Til), and gave Brachy offspring. Anotherwild male (425) produced 7 normals, 2 Brachys and 2 tailless. This
male protably had no tailless gene but had a modifier which shortens
the tail ofTI+, since the six normal F) males, when tested against
Brnchy gave only normal and Brachy offspring. On the other hand,a F) tailless mouse produced by one female (4411) was crossed to
the known tailless line, and to date has produced 4 normal and 16
tailless progeny. This result suggests that this F) tailless was perhaps

RESEARCHES CARRIED OUT IN 1954

15

Table 2. Results of testing mice of wild population Niigata.
~~--_._-

Wild
parentFl
TaillessWildparentFl

NormalBrachy

13
12
5
5

14
9

n-62
n-68

6

Totals from

4 0 6 +1+

35

33

n-63
n-65

14

2

8
10

2

18

0

NormalBrachy

1
6

2
3

7

5

Taillessit

n-61
n-6102
n-64
n-69

4

2 99

Totals from7

Totals from

200 +Itm

or tmlt m

21

Tjt m . It is probable that t m represents a new allele occurring inmolossinus.

Detailed analysis of its genetic behavior is now inprogress.
Wild mice from Niigata-The samples were caught in a barn
at Wano, Nishikambara-gun, Niigata Prefecture. The results oftesting these wild mice are shown in Table 2. Two wild males werediagnosed as +Itm Cor tmjt m). Their tailless offspring were probably
Tjt m , since when crossed with tailless mice of the known balanced~
lethal stock, they never yielded any Brachy young (12 normal and 15
tailless). The F z descendants from one F 1 male include 4 normal and9 tailless. These results seem to indicate that the new t m allele isviable, and gives normal tails when homozygous.
Wild mice from Fukuoka-The samples were sent us throughthe kindness of Dr. Yoshi-Kuni HIRAIWA and Mr. Satoru MINAMI of
the Zoological Laboratory, Faculty of Agriculture, Kyushu University.
One of the two males has to date produced 10 normal, 3 Brachy and16 tailless offspring. The detailed analysis of these tailless mice isin progress.

16

RESEARCHES CARRIED OUT IN 1954

4. Test for Allelism of ' alopecia periodica' and 'furless' in theHouse Mouse (Mus musculus musculus)(by Kiyosi TUTIKAWA)

Alopecia periodica (Ann. Rep. No.3: 9-10, 1953) is a new mutant
which appeared spontaneously in 1951 in an inbred strain of mice
(B 72) kept in this Institute. The mutant is characterized by periodical
shedding of coat hair.
Recently, in 1954, a mutant with a similar phenotype furless wasreported by Dr. E. L. GREEN of Ohio State University. Genetic examination was made by crossing the alopecia mice with the furlessanimals kindly supplied us by Dr. GREEN.
The six litters raised by matings of male furless (fsfs) with female
alopecia (apap ), consisted of 39 normal mice. The reciprocal crosses
gave 32 normals (six litters). In the F2 generation there were 183
normal, 57 alopecia, 59 furless, and 17 double recessive types. Tested
against the 9: 3 : 3 : 1 ratio there is satisfactory agreement between
the observed and expected numbers (X 2=0.624, n=3). This clearly
indicates that ap and fs are due to mutually independent autosomal
genes which are neither allelic nor linked.
The alopecia mice are characterized by thin and lustrous skin; thefurless mice may be distinguished from alopecia mice by having novibrissae at birth. The double recessive type possesses both of thesecharacteristics. Its growth, especially in the stage of the first coatappearance, is much retareded. In some severe cases, these mice weighonly about 1/3 as much as normal mice of the corresponding stage.
The alopecia mice start shedding their coat hair at the age of from13 to 15 days, and at the age of 20-24 days the depilation is usuallycomplete except in some regions, especially around the muzzle. The
furless mice begin to lose their first coat at about the age of 19 days,
the depilation proceeds gradually backward and covers the entire
body within ten days. The double recessive mice show a similarity
to furless mice in the mode of depilation. Detailed observation on
the interaction of ap and fs genes is now in progress.
5. Histological and Cytological Studies of the Testes of Male
Tortoiseshell Cats
(by Takaaki ISHIHARA)
It is known that the male tortoiseshell cat is sterile.

I haveREmARCHEs CARRIED OUT IN 1954

17

studied the testes of five cats of this type. In no case has normal€r:ermatogenesis been observed. The testes contained a few germ
cells besides some SERTOLI'S cells. Most of the spermatogonial cells
showed signs of pycnotic degeneration, and they have never developed
to the spermatocyte stage. In only one individual the testis contained
a few 1st spermatocytes, but these showed some abnormality. In somethere were some spermatogonial cells at the metaphase stage of
mitotic division which was apparently more or less abnormal.

C. CYTOLOGY AND GENETICS OF TUMORS

6. A Sz"mPle Squash Technz"que for Observatz"on of Chromosome
Structures z"n Normal Somatz"c and Malz"gnant Tumor Cells(by Tosihide H. YOSIDA)

Recently, particular attention has been given to cytological study
as an important aspect of cancer researches. In many recently
reported experiments the usual squash technique and staining withacetic orcein, aceto-carmine or aceto-gentian violet are used. This
technique does not show details in the structure of the chromosomes.
The writer has developed a simple squash technique which gives
excellent results in studies of chromosomes in tumor cells as well as
in normal somatic cells. The technique is especially fitted for investigations of chromrsolLes in ascites tumor cells. It consists of two
simple procedures, pretreatment of material with hypotonic RINGER'S
solution and staining with acetic orcein.
(i) A small amount of ascites tumor or a piece of animal tissue
is placed on a slide glass. (ii) Two or three drops of hyponic
RINGER'S solution are immediately applied. The concentration of
RINGER'S solution should vary according to the kind of tumor ortissue: in general, 10 to 20 per cent solution is good for many kindsof material. (iii) After treatment for 10 to 20 minutes, the solution
is removed with blotting paper. (iv) The material is fixed and stainedwith acetic orcein. (v) After 5 to 10 minutes, a cover slip is placed
over the material, light pressure is applied, and the slip is seale:} withparaffin.
Photomicrographs of chromosomes in a male germ cell and tumor
cells examined by using this technique are given in Figs. 1-5.

18

RESEARCHES CARRIED OU'! IN 1954... ~ . . 1M .
i

•

1/.'
.~",
.".~ ~;,, ~·········~······.·····~·a
.· .
~.""

.

•>1

.'

:I

Fig. 1. Chromosomes of a spermatogonial cell of the rat.
Fig. 2. Chromosomes of a YOSHIDA sarcoma celL
Fig. 3. Chromosomes of a tumor cellof the MTK-sarcoma II.
Fig. 4. Chromosomes of a Hirosaki sarcoma
celL
Fig. 5. Chromosomes of a tumor cell of the EHRLICH ascites carcinoma in the mouse.

7. Chromosome Constitutions in Male Germ Cells of the Rat(by Tosihide H. YoSIDA)

Investigations of chromosomes in male germ cells of the rat(Rattus norvegicus) have been carried out by several workers (ALLEN'18, PINCUS '21, PAINTER '28, MINOUCHI '28, OGUMA '35, MAKINO '42,'52). They all record rat chromosomes as having terminal centromeres. GUENIN ('48) analyzed the chromosomes in germ cells of thenewborn rat and found nine pairs of metacentric chromosomes characterized by unequal arms. Recently, MAKINO and Hsu ('54) analyzed
the chromosomes in the embryonic somatic cells of the rat by meansof tissue-culture technique, and demonstrated that eight autosomal

RESEARCHE3 CARRIED OUT IN 1954

pairs (Nos. I, 4, 8, 11, 12,
15, 16 and 19) and the Xelement were two-armed.
The present author examined the chromosomesin the male germ cellsof newborn rats with the13n (U OD "" UO

4 \"

19

«( VII U

00 ""11' U II n U If' VII .K.' ).

UP 011 00 VI} "" OU "" 011 IU VII lCl( leX tt .'" l<" fI, r.

N.

ul " ..

Figs. 1-4. Chromosomes of male germ cells of the rat. Figs. 3, 4.
Serial alignments of the chromosomes. Fig. 3 corresponds with
Fig. 1. Fig. 4 corresponds with Fig. 2.

new squash technique. The results obtained are as follows:
The number of the diploid chromosomes is 42, as recorded by theprevious authors. The serial alignments of these chromosomes show
20 pairs of autosomes (pairs No. 1 to 20), and the X and Y elements(Figs. 1-4). Pairs No.1, 2, 5, 10, 14, 17 and 20 are characterized byhaving subterminal constrictions, with the locus of the centromere
differing in each pair. Pairs No.3, 4, 6, 7, 8, 9, 11 and 15 have terminal
centromeres. Pairs No. 12, 13, 16, 18 and 19 are characterized by having median or submedian centromeres. The elements X and Yarecharacterized by subterminal constrictions. It is a noteworthy factthat the splitting of the chromosomes, which could be observed bymeans of the author's technique, never occurs synchronously in all'Of the chromosomes. As shown in the figures, the splitting of some
autosomes, as well as of the X-element, may be more or less delayed.

8. Characteristics of V-shaped Chromosomes Occurring in Tumor
Cells of the YOSHIDA Sarcoma(by Tosihide H. YOSIDA)
Karyological studies of the YOSHIDA sarcoma cells of the white rat(Rattus norvegicus) have been made by several investigators (MAKINO'51, '52; MAKINO & YOSIDA '49, '51, '52). They have found in these cells
the characteristic V- or J-shaped chromosomes. MAKINO by carefulobservations has confirmed the presence of stem-cells of the tumor

20

RESEARCHES CARRIED OU'J. IN 1954

which are characterized by subdiploid chromosome complex (about 40
in numbers). This complex consists of 22 to 24 rod-shaped elements
and 16 to 18 V-shaped elements including one particularly large Vshaped element.
I have examined manytumor cells with the aid of
the new squash technique,and found that the majorityof the tumor cells of theu~o r", 19 uu «t ex .. .VV I I . . . . . ."., , .

'v, ,-, U VI II U" .. U

II U ft ..

t.

Fig. 1-4. Chromosomes of YOSHIDA sarcoma cells.
Figs. 3, 4.
Serial alignments of these chromosomes.
Fig. 3 correspondswith Fig. 1.
Fig. 4 corresponds with Fig. 2.

YOSHIDA sarcoma have 40 chromosomes including two large V·shaped
elements. These two V·elements slightly differ in size from eachother. The two arms in each element show a slight difference insize, moreover they often differ in the manner of splitting. From theseobservations it has been surmised that the two arms of the V·shaped
elements are non-homologous and that these chromosomes have originated by terminal fusion of the non-homologous, rod· shaped chromosomes indicated as Nos. 6 and 7, and Nos. 8 and 9 in Figs. 1-4. All
the other chromosomes exactly corresponed in their morphologicalcharacteristics to those in the normal somatic cells.

9. Nature of V-shaped Chromosomes in Tumor Cells of MTKSarcomata II and III(by Tosihide H. YOSIDA)
The MTK-sarcomata II and III represent two different strains of atransplantable ascites tumor which were induced by the applicationof azo dyes (TANAKA and KANO 1951, 1952, UMETANI 1954). According
to MAKINO and KANO (1953), the tumor cells of the MTK-sarcoma II
have a well-balanced complex of subdiploid chromosomes, 40 orthereabouts. A morphological analysis of the chromosomes, hasrevealed that the chromosome complex consists of two distinct

21

RESEARCHES CARRIED OUT IN 1954

groups, one containing rod-shapei chromosomes and the other comprising V- and J-shaped elements. Among the latter group, the solitary
large V-shaped chromosome is very prominent.
The chromosomes of tumor cells of MTK-sarcoma III have been
observed .by UMETANI (1954). According to him the chromosome
number of MTK-earcoma III varies widely from 26 to 82, with 40 as
the mode. This tumor is characterized by having two strains oftumor stem-cells, of which one is characterized by having a prominent
V-shaped element, while this element is missing in the cells of theother strain.
The present author investigated the chromosomes of MTK-sarcomata
II and III with the new squash technique, and obtained the following
results.
i) MTK-sarcomaII: Close microscopical examination of manytumor cells has revealed that most of the tumor cells contain 40~~ ~ f~ p~hromosomes, indud~!i:;} ~ ~V "~.r v(,~.;
mg two large V-shaped
...~.
'(.~
Jr. <&• ..~_ \ elements. These ele~-, ~:~'In#tl4''\(\.:~~.. ments are similar in~.. ..=..... iF/"'lIt~/I ~ 'I. U \size and shape to those~ /i,v 1
"2

..

3

4

0 b(~ n!ll~«f~Dn UnBt Ut 1('
11.II I»)" It ro )v_' .. " n :rI' "II U ut. ; ;'Xy

II If .. If •• f.

Figs. 1, 3. Chromosomes of a tumor cell of the MTK-sarcoma II.
Figs. 2, 4. Chromosomes of a tumor cell of the MTK-sarcoma
III.
Figs. 3, 4. Serial alignments of the chromosomes.
Fig. 3
corresponds with Fig. 1.
Fig. 4 corresponds with Fig. 2.

found in the YOSHIDA sarcoma cells. By comparing the chromosome
set in the normal somatic cells with that in the MTK-sarcoma II cell,it has been concluded that both of the two large V-shaped elements
probably arose by terminal fusion of rod-shaped chromosomes, one
probably by fusion of No.6 and No.7, and the other by fusion of No.
8 and No.9 (Figs. 1 and 3). This finding exactly corresponds withthat made in the YOSHIDA sarcoma cells. A striking difference between the chromosome complexes of YOSHIDA sarcoma and MTKsarcoma II may be noticed in the sex chromosomes. The former

22.

RESEARCHES CARRIED OUT IN 1954

sarcoma originated in the male rat, and consequently has X- and Ychromosome sets, while the latter was derived from a female rat,and has two X-chromosomes.
ii) MTK-sarcoma III: The morphological characteristics ofthe chromosomes in this tumor nearly correspond with those of YoSHIDA sarcoma and MTK-sarcoma II. The number of chromosomes inmany tumor cells was 40. The tumor cells usually include two large
V-shaped elements. By comparing the chromosome sets of MTK-sar·coma III with those of the normal somatic cells, YOSHIDA sarcoma and
MTK-sarcoma II, it has been revealed that one of these elements wasprobably derived from the terminal fusion of chromosomes No. 7
and No.8, and the other from the fusion of the other member of No.
8 chromosomes and one No.9 chromosome. This sarcoma includes
the X- and Y- chromosomes. The remaining chromosomes exactly corresponded with those found in the normal soma, YOSHIDA sarcoma andthe MTK-sarcoma II.
10. Karyological Characteristics of Tumor Cells of the Hirosaki
Sarcoma(by Tosihide H. YOSIDA)

This sarcoma was established by Dr. USUBUCHI of the Hirosaki
Medicai College. and seems to have been derived from a spontaneous
epithelial tumor in a white rat (Rattus norvegicus). Observation of
the chromosomes in this tumor have been carried out by MAKINO and
KANa (1953), and KANa (1954). According to MAKINO and KANa
(1953), the tumor cell possesses about 40 chromosomes, including
certain numbers of rod-, v- and J-shaped elements of varying sizes.
The cell is also characterized by having, without exception, prominent
V-shaped chromosomes which vary in number from one to five.
Microscopical observations on many metaphase plates using the
new squash technique have revealed that cell having 38 chromosomes
occur most commonly. Among these chromosomes, there are usuallyfour large V-shaped elements. Closer examination of these chromosomes has shown that all of them are of a composite nature, andseem to have originated by fusion of two original chromosomes in
the following manner:

23

RESEARCHES CARRIED OUT IN 1954

Composite chromosomes

Probable components

I
II
III

2a + 6a6b + 17a
9a + lla
9b + 15aIV

One of these composite chromosomes was probably derived fromthe fusion of one No. 2 chromosome and one No. 6 chromosome; thesecond composite chromosome was probably derived from the fusionof the other member of the No.6 chromosome pair and one No. 17
chromosome; the third composite chromosome, from the fusion of
one No. 9 and one No. 11 chromosome; and the fourth composite,from the fusion of the other member of the No.9 chromosome pairand one No. 15 chromosome (Figs. 1-4).
Another noteworthy fact
about this sarcoma is thata very small dot-like element usually occurs in its
chromosome complex. Thiselement is similar in size

H() (( "Jf I n ,U J1 11 "It
4 () ,.., U 0& .. J "Of tt..u, nu."

3

Q

II It 9 Ie u l '
XI

II I

un .. , I •..

Figs. 1-4. Chromosomes of Hirosaki sarcoma cells.
Figs. 3, 4.
Serial alignments of the chromosomes.
Fig. 3 correspondswith Fig. 1.
Fig. 4 corresponds with Fig. 2.

and shape to the small knob of the No.2 chromosome, and it ispossible that the former originated by the detachment of the small
knob of the latter.

11. A Further Study on the Transplantability of MY-mouse 1
Sarcoma(by Tosihide H. YOSIDA and Takaaki ISHIHARA)

The MY-mouse sarcoma is a kind of spindle cell sarcoma. It wasdeveloped in a So-strain mouse, which has not yet been established i

RESEARCHES CARRIED bUT IN 1954
as a pure inbred strain. It has been shown that the transplantabilityof this tumor shows considerable difference according to the strain of
the host used (YosIDA & ISHIHARA 1953). In our transplantation experiments, we found that the mice of D-strain showed a high transplantability. By selection of these mice we have succeeded in establishing a strain (D.103) showing a very high transplantability (92%).
On the other hand, C3H strain mice commonly showed negative
results regarding the transplantability of this tumor (YosIDA &ISHIHARA 1953). The transplantability was examined in the F 1 hybrids
between the two strains CD-103xC3H). In all of them the transplantation was successful. Accordingly, it was assumed that thetransplantability of this tumor was controlled by some dominant
genes (H-genes). To decide the number of these genes, the transplantability was examined in the backcross C3HxFl; 25.72 per centof the RF1 hybrids showed a positive transplantability. Based on theabove results, it has been surmised that about two dominant H-genes
are concerned with the transplantability of the MY-mouse sarcoma.
It is noteworthy that 'the transplantability of this tumor to theS-strain has been recorded as high as 72.35 per cent CYasIDA &ISHIHARA 1954). But the F 1 hybrid (SxC3H) usually shows negative
results with respect to transplantation.

12. Development of Resistance to Nz'trogen Mustard N-oxide in the
EHRLICH Ascites Carcinoma(by Takaaki ISHIHARA and Tosihide H. YasIDA)

It is important from the medical and biological standpoints to investigate the mechanism of development of resistance against anticancer agents. In order to contribute something to this problem, we
have experimented with the EHRLICH ascites carcinoma as tumor
material, and nitrogen mustard N-oxide (methyl-bis-(,8-chloro-ethyl)lamine-N-oxide) as anticancer agent.
The method was as follows: 5 x 106 of the EHRLICH ascites carcinoma
cells were immersed in a solution (0.64 mg/ml) of nitrogen mustard
N-oxide in vitro for one hour at 20°C. The treated tumor cells were
injected into the intraperitoneal cavities of mice. When the trans·
plantation showed a positive reaction, the tumor cells were again
treated with the same solution in the same way as before; thisprocedure was repeated two or three times. As the result of thisRESEARCHES CARRIED OUT IN 1954

25

treatment, we obtained two lines (SL-6 and RL-l) resistant against thischemical. Moreover, we were able to establish a resistant line (RL-2)
from the untreated control tumor. By intraperitoneal injections of
the chemical (0.32 mg/mouse), a marked reduction and disintegrationof tumor cells occurred in the untreated control line, while in theresistant lines the reduction in the number of tumor cells was verysmall. After five successive treatments it appeared as if the tumor
cells had developed a resistance to the lethal amount of this chemical,
and SL-6 was established as the line resistant to the highest dosage.
To determine something of the nature of this resistance of tumor
cells against the chemical, the following experiment was carried out.
Some chemical such as ethoxy-nitrog~n mustard N-oxide, urethane,colchicine, acriflavine or H2 0 2 was injected into the animal bearing atumor, in which the resistance to nitrogen mustard N-oxide had been
established. The tumor cells showed a sensitive reaction to suchchemicals as urethane, colchicine, acriflavine and H 20 2, while they wereresistant to ethoxy-nitrogen mustard N-oxide. The tumor cells of thenormal control line were very sensitive to all the chemicals used.
B:ised on the above experiment, it may be concluded that theresistance of tumor cells against nitrogen mustard N-oxide is independent of the resistance against the other chemicals.

13. Two Spontaneous Tumors Occurring in Inbred Rats
(by Tosihide H. YOSIDA and Takaaki ISHIHARA)

Both of these tumors spontaneously developed in the inbred ratsof two pure strains W- and Wyne pink-eyed yellow reared in ourlaboratory. This report deals with the histological characters of thesetumors and the results of some transplantation experiments.
i) Tumor in W-strain: A female of ~train W-545 developed aperitoneal tumor in a lymph-gland of th':l mesentery. Histologicalobservation revealed that this tumor was a reticulum cell sarcoma.
It was transplanted into many rats, with negative results.
ii) Tumor in Wyne-pink eyed yellow rat: In this case thetumor developed on the left leg in a male. The histotogical study
reveale-i it to be a kind of fibrosarcoma, which is characterized byshort spindle cells. It was transplanted into six individuals of thestrain in which it arose. All of them yielded positive results. By
repeated transplantation experiments, this tumor was established as

26

RESEARCHES CARRIED OUT IN 1954

a transplantable strain of fibrosarcoma.
The authors wish to express their gratitude to Prof. Kunio OOTA
of the Cancer Institute of Tokyo for his important criticism of thehistological characters of these tumors.

D. GENETICS OF POULTRY
14. A Sex-linked Nervous Disorder in the Domestic Fowl
(by Takatada KAWAHARA)

In a breeding stock hatched for replacement purposes in the springof 1954, there were found some individuals afflicted with a nervous. disorder characterize:! by a tremor of small amplitude of head andneck. The examination of the pedigree records has disclosed that allthese individuals were derived from a single mating of a Barred
Plymouth Rock sire SG2-125 and his half-sib 307. This nervous dissorder usually appears in chicks as young as one day old, but theappearance may be delayed to four weeks or more in some individuals.
About half of the afflicted females are unable to stand up or walkwhen they reach the age of twenty weeks, and they eventually die.
The other half reach sexual maturity, but often give poorer egg production than the normal pullets. As far as I know, five hereditary
nervous disorders have been reported in the domestic fowl. Of thesethe "Shaker" described by SCOTT et al. (1950) seems to be very
similar to the present case in symptoms of affliction. The two strains
are, however, somewhat different in lethality and in histological characters of the nervous system. Two heterozygous and phenotypicallynormal males were mated with normal females and gave 21.1 % afflicted females, the male offspring all being normal. The mating of F 1
afflicted females with the heterozygous males yielded 55.5% (females
24.4%, males 31.1%) afflicted chicks. The F r afflicted females matedwith homozygous normal males produced no afflicted chicks. These
breeding results indicate that this nervous disorder is- due to a sexlinked recessive gene. Comparative histological examination of thetissues, particularly of the central nervous system, was made on the
afflicted and normal chicks. The tissues were fixed in BOUIN's or
MULLER'S fluid, and stained with haematoxylin and eosin. In the cerebellum of the abnormal fowl, signs of degeneration of the PURKINJE
cells, which are manifested first in the gyri region of cerebellum have

RESEARCHES CARRIED OUT IN 1954

27

been observed. According to SCOTT et al., in the "Shaker" breed the
PURKINJE cells in the gyri regions degenerate later than those inother portions in the cerebellum.
15. Genetic Variation and Covariation in Economic Traits in Some

Breeds of Chickens(by Yukio YAMADA)

Estimations for heritability and genetic correlations were made fromdata of three years' records of some breeds of chickens, i.e. W.
Leghorns, B. P. Rocks and R. 1. Reds including 783 trapnested progeny
which were sired by 57 males to 253 females. The birds studied werelimited- to those-which had records for all traits to be ,analyzed in thisstudy. The method used for estimation was based on combined sireand dam components of variance and covariance obtained from thepooled analysis within breed and year.
.
The traits investigated were sexual maturity, body weight and rateof egg production. Measurements of body weight were taken at theage of sexual maturity and at the age of about nine months. The rateof egg production in percent was measured for three periods, i.e. thewinter rate from December 1 to the end of February, the spring ratefrom March 1 to the end of May and the total rate from the first egg
to the end of May.
The heritability for sexual maturity'has been estimated to be 0.480.
This value is higher than the values so far obtained by other investigators, except the 0.522 reported by KING and HENDERSON (1954) assuming hatch effect. No maternal effect was found.
The estimate of heritability for body weight is 0.462, which agrees
fairly well with the values reported by other investigators. The bodyweight at the first egg was estimated to have a heritability of 0.428.
Heritabilities for the rates of egg production have been found to be
0.313, 0.169 and 0.148 for winter, spring and total rate, respectively.
WYATT (1954) reported the corresponding values to be 0.41, 0.38 and
0.51, respectively, on the basis of combined sire and dam components
of variance, and also 0,11, 0.00, and 0.00, respectively, based on regression of offspring on parent. LERNER and CRUDEN (1948) reported theheritability of accumulative monthly egg production, in which an estimate of 0.145 was found for egg production in "production line" tothe end of May; and WILSON (1948) obtained the value of 0.31 forproduction rate. Although the traits studied by them are not strictly

28

RESEARCHES CARRIED OUT IN 1954

comparable with mine, the present estimates may be considered asfairly valid for production rate.
The genetic correlations involving sexual maturity, body weight andproduction rate have been estimated as follows: 0.003 for sexual
maturity and mature body weight; -0.217 for sexual maturity and bodyweight at sexual maturity; -0.210 for sexual maturity and winter production rate; 0.173 for the winter production rate and body weight;
0.187 for winter rate and spring rate; 0.817 for spring rate and total
rate; and 0.767 for body weight at sexual maturity and mature bodyweight.
These estimates of genetic correlations may be subject to rather
large sampling errors, and further estimates based on the regression
method are to be made. Further discussion and criticism will be attempted after additional information has been obtained.

E. GENETICS AND BIOCHEMISTRY OF SILKWORM
AND OTHER INSECTS
16. "Small Egg ", a Mutant in the Sz"lkworm(by Yoshimaro TANAKA)"Small egg" is a recessive mutant which appeared in F 2 of thecross between a hereditary mosaic strain and a Chinese pure race, bothi' of which strains had been kept under my own care for many years.
The mutant gene is designated as sm.
The sm egg is smaller both in length and in breadth by about 20%
than the normal egg of the same family. The sm/sm moths lay small
eggs only, while +/sm females give batches of normal eggs only. All
small eggs are unfertilized, hence we cannot breed a pure sm/sm strain.
1) Lethality of sm-gene: In the progeny produced by interbreeding +/sm individuals, the normal and small eggs segregate, but
not exactly in a ratio of 3: 1, the small egg type coming always shortof expectation. There were, for example, 397 normal egg batches and85 small egg batches among the total 482. From this and anothersources, the lethality of sm/sm zygotes is calculated as about 35%.
The lethal action seems to occur not in the embryonal, but in thelarval and pupal stages, because no particularly large numbers of deadeggs are observed in segregating egg batches. This lethality must be
taken into consideration when the phenotypic ratio is compared to thetheoretical ratio.

RESEARCHES CARRIED OUT IN 1954

292) Linkage of genes sm and Ze: The existence of linkage between sm and Ze (zebra marking in Chromosome III) was proved by
the results of various matings, i.e. Ze sm/ + + 9 X + + I + sm 0, Ze+ 1+ sm 9 X + +/ + sm O. + sm/ + + 9 x Ze sml + + 0 and + sml+ + 9 X Ze +1+ sm O.
3) Crossingover between sm and Ze: As is well known,
crossingover takes place only in the male sex in Bombyx, so the crossingover values in the present case can be calculated exclusively fromthe data by the matings + sm 1+ + 9 x Ze sm 1+ + 0 (repulsion
phase) and + sm/ + + 9 x Ze +1+ sm (coupling phase), sm/sm females
being perfectly sterile.
The crossingover value obtained from the repulsion matings was
21.03%. while that calculated from coupling matings was 18.03%. The
repulsion matings included as many as 5,974 batches (females) in total,
while the coupling matings were' only 893. For this and some otherreasons. the figure 21.03% seems to be more reliable than the other.
17.

Effect of Temperature in the Larval Stage on the Development of Multilunar and Multistar Markz'ngs(by Mizuo YOSHIDA and Yoshimaro TANAKA)

A striking effect of incubation temperature on the size and numberof brown spots composing themultilunar marking was reported by
T. HIROBE (1951). Y. TANAKA (1954) observed similar results in themultilunnar as well as in multistar marking. The present experiment
was undertaken to determine whether there is any influence of therearing temperature on the development of the markings in question.
Three batches of multilunar strain and two batches of multistar
strain were incubated at an intermediate temperature C20°C). Immediately before hatching, each egg-batch was divided into two halves,of which one was reared at a high temperature (25°C), and the otherat a lower temperature (15°C). The markings in the fourth and fifth
instars were carefully observed.
The result was distinctly negative; i.e. no trace of influence of therearing temperature was found. We have come to the conclusion.
therefore, that the distribution types of the above-mentioned markings,
though they fully develop first in later instars, are determined before
hatching, so as to be entirely immune to .further environmental
changes.

30

RESEARCHES CARRIED OUT IN 1954

18. Genetical and Biochemical Studies on Yellow Lethal Silkworm Larvae. III. On the Property of Phenol Oxidase in thisMutant.
(by Mitsuo TSUJITA and Bungo SAKAGUCHI)
It is known that the embryos homozygous for the gene lem l develop
normally to the youngest larval stage and die immediately after the
first moulting. We have been interested in the direct cause of thisdeath, which is apparently due to imperfect differentiation of themandibular cuticle and incomplete hardening of the cuticlar layer ofthe hypodermis. In a previous report, the maternal inheritance of thisstrain, and also biochemical studies of the larvae, with special reference
to the difference in the nature of pterin and some other chemical
substances contained in the normal and mutant larvae, were recorded.
Further biochemical studies with the normal and the mutant strains
were carried out, and the following results were obtained.
1) The phenol oxidase activity of the epidermal tissue in thelarvae directly after the first moulting of the normal, lem and leml
strains was measured by using a WARBURG manometer. A homogenateof the tissue in isotonic sucrose solution was prepared as the crude
enzyme solution, and tyrosine or dopa was used as substrate. No difference in the activity of tyrosinase was detected among these strains,
but the dopa oxidase activity in both the normal and /em larvae wasstronger than that in the /em l larvae.
2) The next question is whether or not the weak dopa oxidase
activity in the epidermal tissue of the /em l is due to a smaller quantityof the oxidase, or to the presence of some inhibitor of this enzyme.
In order to ascertain this point, the following experiments were carriedout. (i) The two crude enzyme solutions obtained by the above-mentioned procedure from the epidermal tissues of the + and /em, weremixed in the ratio 5: 5, 7: 3 or 3: 7, and the dopa oxidase activity ofthese mixed enzyme solutions was measured by means of a WARBURG
manometer. In these experiment, the enzyme activity is given by themean of the activites of the two enzyme solutions. (ii) In order tomake clear whether an inhibitor of dopa oxidase is present in the /em lor not, a solution was extracted by phospate buffer solution (pH 6.4)
from the homogenate of the first moulting larvae of the /em l , andthe solution was precipitated by saturated ammonium sulfate, and thenthe supernatant solution was filtered through WHATMAN No. 42 filter
paper to remove the activity. The solution obtained by such a pro-RESEARCHES CARRIED OUT IN 1954

31

cedure was added to the crude enzyme solution extracted from thenormal larvae, and the dopa oxidase activity was measured by thesame procedure as that previously mentioned. The result shows thatthe enzyme activity was not inhibited by the addition of the solution.
(iii) Therefore, it seems that the low activity of dopa oxidase in theepidermal tissue of [em' is not due to the presence of an inhibitor, butthat it is directly controlled by the gene [em' itself.
3) Some + and [em' larvae in the first moulting stage were
homogenated and dividid into pigmental and residual fractions by theaddition of an acidified methyl alcohol, and the metals contained ineach of the fractions were traced by paper chromatography. Fe andCu were found in the pigment fraction of the +, while Ni and
Ti were detected in the fraction of the [em'.
4) In order to know the effects of these metals on the dopa
oxidase activity, metal ions of Cu, Fe, Ni and Ti were added to crude
enzyme solution prepared from the larval epidermal tissue of thenormal strain, and its enzyme activity was measured by the W ARBURG
manometer, by using dopa as substrate. The results have shown thatCu and Fe accelerate the enzyme activity, but Ni and Ti do not.
5) In order to ascertain the relation of xanthopterin-B obtained
from the larval epidermal tissue to the dopa oxidase activity, an experiment was carried out by using as crude enzyme solution thehomogenate prepared from the epidermal tissue of the normal
strain. The dopa oxidase activity of the preparation, containing bothxanthopterin-B and the crude enzyme solution, was measured by amanometer. No effect of xanthopterin-B on the dopa oxidase activity
was found.
6) Based on these experimental results, the following hypothesis
is proposed. The epidermal tissue of the [em' can adsorb Ni and Ti,
and the enzymes connected with these metals are closely related to
the formation of xanthopterin-B. The tissue, however, shows only aslight tendency of adsorbing Cu which acts as an active center oftyrosinase, which in turn plays an important role in melanin formation, and this results in the production of a very small amount ofmelanin pigment.

32

RESEARCHES CARRIED OUT IN 1954
19. Comparative Studz"es of Yellow Lethal Lemon Larvae ([em l )and Albt"no Lethal Larvae (al) of the Sz"lkworm from the Vz"ewpoz"nt of Bz"ochemical Genetics(by Mitsuo TSUJlTA and Bungo SAKAGUCHI)In both the lem E and the al larvae, the mandibular cuticle does
not develop properly, so that they cannot chew mulberry leaves, andstarve to death directly after the first moulting. The lethal lemon
larva contains in its epidermis a large amount of yellowish pigment
which gives the body a yellowish color, while the lethal albino larva
has a light brown color.
Comparative biochemical studies of these two lethal strains werecarried out. The results may be summerized as follows:
1) Pterin. Extractions were obtained from the +, lem, lem E andal larvae after the first moulting by N/2 ammonia, 80% methanol orwater. Paper chromotagraphic analysis of the extracts was carriedout for the detection of the pterin. Butanol acetic acid, 4% phenol or
3% Na·citrate were used as the developmental solvent of the analysis.
Leucopterin, isoxanthopterin and riboflavin were detected in the allarvae, and xanthopterin-B in addition to these three substances wasfound in the lem E larvae; the relative quantities of isoxanthopterin inthese strains, have been shown to be + > al >lemE•2) Comparison of phenol oxidase activity. By using a WARBURG
manometer, the phenol oxidase activity in the epidermal tissue of thelarvae directly after the first moulting of the four strains, +, lem,lem E and al, was measured. The homogenate of the larval epidermal
tissue (homogenized in isotonic sucrose solution) was used as crude
enzyme solution. The four substrates, tyrosine, p-cresol, dopa andpyrocatechol were used. When tyrosine and p-cresol were used as
substrates, no difference in the phenol oxidase activity could be detected among the three strains. However, when dopa and pyrocatechol
were used as substrates, the enzyme activity in + and lem was foundto be somewhat stronger than in lem E• On the other hand, the phenol
oxidase activity was weaker in al than in +, lem or lem E, when all
the four substrates mentioned above were simultaneously used. Thistendency was especially clear in the case of the two substrates, dopa
and pyrocatechol.
3) Comparison of cytochrome oxidase activity. The cytochromeoxidase activity in +, lem E, and al larvae directly after the first moult-RESEARCHES CARRIED OUT IN 195433

ing was measured by SCHNEIDER and POTTER'S procedure (1943). A
homogenate of the tissue was prepared as the crude enzyme solution,and cytochrome c was used as the substrate. The oxidase activiiy
was stronger in the normal strain than in lem1 or al larvae, but. no
difference was found between lem1 and al in this respect.
4) From these findings, the following conclusion may be deduced:
Although the lethal lemon and the lethal albino larvae show somephenotypical resemblance, they are different in their metabolic physiology. The gene lem 1 is mainly responsible for the abnormal pterin
formation, while the gene al has a smaller capaGity of melanin formation. These physiological abnormalities have ~econdary effects uponother metabolic processes, and produce somewhat similar phenotypic
expressions.

20. Biochemz'cal Studies on the Hibernatz'ng Character z'n the
PilOtoperiodism of a Wild Silkworm, Antheraea pernyz'(by Bungo SAKAGUCHI)TANAKA ('41-'43, '50-'51) has reported that photoperiodism plays themost important role in determining the hibernating character of theChinese tussar silkworm, Antheraea pernyi GUER. In order to makeclear the biochemical background of this mechanism, the present experiments were undertaken. The results of the experiments may besummarized as follows:
1) Artificial control of the hibernating character has been attemptedby injection of various chemical substances or by transplanting variousorgans into nonhibernating pupae. It has been found that blood,urine, brain or corpora allata of the pupae, or a mixed solution ofsubstances of the citric acid cycle, Na-succinate or cytochrome c, areapparently effective in changing hibernating pupae to nonhibernating.
2) Fluorescent substances in the blood and urine of the hibernating and nonhibernating pupae were analyzed by paper chromatography.
Greenish yellow, yellow and blue fluorescent substances were foundin both of these pupae. In the nonhibernating pupae substances ofintense purple and purplish blue color were detected in addition to thethree substances mentioned above. Both these two substances seemto have a photo-decomposible character.
3) The cytochrome oxidase activity of the epidermal muscle was

34

RESEARCHES CARRIED OUT IN 1954

measured by the WARBURG manometer. The enzyme activity in nonhibernating pupae was markedly stronger than in the hibernating ones.
The activity of the enzyme was accelerated by the injection of urine
and fluorescent substances extracted from the urine of nonhibernating
pupae.
4) Enzymatic activity belonging to the citric acid cycle and appearing in epidermal tissue of the hibernating and nonhibernating pupae
was tested by the modificatory method of GREEN ('50). The activityof each enzyme taking part in the cycle of the nonhibernating pupae
was greater than in the hibernating pupae.
5) It may be surmised from these findings that the activity ofcytochrome oxidase of the hibernating pupae may be activated byfluorescent substances in the urine and blood of nonhibernating pupae,
and in turn a group of enzymes belonging to the citric acid cycle are
activated.
Some of the fluorescent substances in blood and urine of the nonhibernating pupae are known to be decomposed by light. This fact
seems to explain at least in part the close relationship between thehibernating character and photo-periodicity.

21. Biochemical and Genetical Studies on Wild Silkworm. II. On
the Nature of the Pigments in the Epidermal Tissues of the
Chznese Tussar Silkworm, Antheraea pernyi(By Bungo SAKAGUCHI)

The chemical nature of the pigments in the epidermal tissue of theEri·silkworm has been reported by the author (SAKAGUCHI 1952). In
this work, the pigments in epidermal tissue of the Chinese tussar silkworm, in Eri-silkworm and in the silkworm are compared from thebiochemical and genetical view points.
Detection of carotenoid pigments: The pigments were extracted with a mixture of petroleum ether and benzene from thehypodermal tissue of full-grown larvae. Several kinds of pigment in
the extracts were separated by partition chromatography through acolumn of alumina, and then yellow, orange-yellow, orange, purplish
fluorescent substances and a few other pigments were isolated. The
pigments, except the fluorescent substances, were easily oxidizable andshowed a peculiar reaction against H 2S04, HCI or ZnCI2 • Moreover, theRESEARCHES CARRIED OUT IN 1954

35

adsorption band showed three peaks between 400 and 500 m,.". Thesefindings indicate that the pigments are composed of xanthophyll andcarotene, which are found also in epidermal tissue of the Eri-silkworm
and in the yellow cocoon of the silkworm.
Detection of fluorescent substances: The fluorescent sub.
stances were extracted by solvents, acidified ethanol or hot water,from the epidermal tissue of the full-grown larvae, and the extracts
were analyzed by paper chromatography. In the larval epidemral
tissue of the Chinese tussar silkworm, the same leucopterin and isoxanthopterin as those found in the silkworm and the Eri-silkworm weredetected.
Permeability of the larval epidermal cells: The larvae of
the Chinese tussar silkworm, of the Eri-silkworm and of the silkworm
were fed with oak leaves painted with various synthetic pigments. It
has been found that toluidine blue, neutral red and methylene blue
can be adsorbed in the epidermal cells, and the hypodermis takes thesecolors. The pigments adsorbed in the cells in the posterior part ofthe body of the Chinese tussar silkworm, vanished after some time.
Conclusion: It seems clear from the above experimental resultsthat among the three species of silkworms there are some differencesin the carotenoid pigments and fluorescent substances contained intheir epidermal cells as well as in the permeability of the pigments
through the hypodermis. The synthesis of fluorescent substances andpermeability of carotenoid pigment are probably controlled by geneaction. The facts found in this experiment suggest some interesting
problems regarding the differences in these physiological properties
among the three silkworm species.

22. Pterins found in the Silkworm(by Saburo NAWA)

It has been confirmed that the purple fluorescent snbstance detectedin egg and larval epidermis in the normal strain of the silkworm isisoxanthopterin.1•2)
The epidermis of the mutant lemon Clem) of the silkworm contains
a yellow pigment which has been named xanthopterin-B31. We41 haveobtained 2-amino.4-hydroxypteridine-6-carboxylic acid through photodecomposition of this pigment. This shows beyond any doubt that
xanthopterin-B is a derivative· of 2·amino-4-hydroxypteridine.

S6

RESEARCHES CARRIED OUT IN 1954

These observations suggest that an abnormal metabolism of pteridine
compounds exists in the mutant lemon.
It has been detected that the yellow pigment in the head of themutant strains, se and cl of Drosophila melanogaster, is identical withxanthopterin-B found in lem of the silkworm. Recently, FORREST et al.S)
proposed the structure 2-amino-4-hydroxy-7, 8-dihydro-8-lactylpteridine6-carboxylic acid for the yellow pigment.
1) HIRATA. Y. and NAWA, S. 1951. Compt. rend. soc. bioI. 145: 651.
2) NAWA. S. et at. 1954. J. Biochem. 41: 657.
3) HIRATA, Y., NAKANISHI,
K., and KIKKAWA. H. 1950. Bull. Chern. Soc. Japan 23: 76.
4) NAWA, S.
and TAIRA, T. 1954. Proc. Japan Acad. 30: 632.
5) FORREST, H. S. and
MITCHELL, H. K. 1954. J. Am. Chern. Soc. 76: 5658.

23. The Relationship between Eye Pigment and Pterins ofDrosoph£la melanogasterV(by Saburo NAWA and Toshifurni TAIRA)
The pterins found in D. melanogaster were investigated. The
relative amount of isoxanthopterin contained in the whole body ofthe males of various mutants of D. m. separated by paper chromatography was measured by the fluorometric accessory set of a BECKMAN
spectrophotometer.
The males of the age from 15 days after emergence were used asmaterial. By the agreement in Rf value, fluorescence, ultraviolet absorption spectra and chemical nature, it has been confirmed that thefluorescent substances obtained from the material consist mainly of
2-amino-4,7-dihydroxypteridine (isoxanthopterin) and the derivativesof 2-amino·4-hydroxypteridine. The relative amounts of isoxanthopterin in these mutants are as follows :-v, 54; en, 48; wild type, 43; .
pr, 43; dke, 35; st, 35; car, 35; cl, 32; ea, 27; PP, 17; em, 12; wa , 4;rb, 4; bw, wand v: bw, O.
It has been found also that the yellow eye pigment contained in
the se and cl mutants is a 2-amino-4-hydroxypteridine derivative andidentical with xantopterin-B found in the mutant "lemon" of thesilkworm.
The strains of D. m. without any red eye pigment, like bw, wandits alleles, have no pteridine compounds while the strains having redor yellow eye pigment, such as wild type, en, v, se and cl, etc.,

37

RE3EARCHE3 CARRIED OUT IN 1954

contain pteridine derivatives in large quantities.
These data suggest that there is a close relationship between thered or yellow eye pigment and the pterins.
1)

NAWA, S. and TAIRA, T. 1954. Proe. Japan Aead. 30: 632.

F. POPULATION GENETICS OF SOME INSECTS
AND A LAND SNAIL
24. Polymorphz"sm Found z"n Some bzsects and z"n a Land Snaz"l(by Taku KaMAl)a) The lady- beetle Harmonz"a. Some new population samples
were obtained during the current year. The comparative study ofa new sample from Suwa with older samples from the same locality
has yielded some rather interesting results. The oldest sample fromthis locality dates back to 1912 while the newest one was obtainedin the winter of 1954. Between these two extreme years, samples weretaken in 1913, 1914, 1915, 1917, 1920, 1930, 1942, 1943 and 1950. It has
been found by examining these samples, that a significant change inthe composition of the population occurred during the period 1920-1950
which covers probably about 120 generations of the beetle. This change
is perceived as a steady decrease of the red form (sucdnea) which is dueto the recessive gene, and a concomitant increase of the darkest form
(conspz"cua) which is due to the gene at the highest peak of dominance.
The possibility of the effect of immigration from the adjacent colony
has been ruled out by the examination of the ratio of the individuals
provided with a ridge on the elytra. The presence or absence of
this ridge is determined by a pair of mendelian genes, and the genefor presence is dominant over the gene for absence. The incidenceof the gene in the colony shows a regular geographic gradient along
the longitudinal axis of the Japanese island chain. It has been foundthat the incidence of the gene in the Suwapopulation has remained
unchanged from 1920 through 1954.
This observation makes it almost certain that the regular change
in the proportion of individuals with different elytral markings in the
Suwa population was caused by selection. Based on this idea, thecoefficient (s) of selection of the recessive form was calculated andthe following results w~re obtained:

38

RESEARCHES CARRIED OUT IN 1954

PeriodNumber of generations

1920-193040

1980-1942, '43

50s0006400.00647

1942, '43-1950

30

0.00706

Thus, a rather uniform value 0.0064-0.00706 was found for s. From
this valve of s, the rate of change in the incidence of the recessive
gene .Jq has been calculated, and the value 0.00093-0.00098 was
obtained. It is to be noted that this value is too great, probably 100
times greater than what can be expected for any change due tonatural mutation, and this last possibility is also excluded from thecause of this regular change in the composition of the population.
My attempt to correlated this change to some regular change in the
climatic condition in Suwa during the same period has been unfruitfulso far, although the presence of such a correlation is most likely.
At any rate, this finding seems to be interesting to the students ofevolution in the sense that an actual instance of microevolutionarychange in a natural population of an insect was witnessed during
the life time of a man, and its process was followed, and its tempo
and grade were numerically expressed in terms of gene incidence.
Reference: KOMAI, T. 1954. An actual instance of microevolution
observed in an insect population. Proc. Japan Acad. 30: 970-975.

b) The lycaenid butterfly Neozephyrus taxi/a. The fifteenth population sample of this polymorphic butterfly was obtained
from Nose in Osaka Prefecture. This sample contained the four
different types of females, 0 (uniform brown). A (with orange
markings), B (with bluish suffusion) and AB (with both orange
markings and bluish suffusion) in the following proportion:

o

AB

Type
Number

106

31

99

26

262

%

40.46

11.83

37.79

9.92

100.00P=0.1l6

q=0.277AB

r=0.636

D= 1-1.029= - 0.029

Total

p+q+r=1.029
uD=0.0097

If adjustment is made so as to make p'+q'+r = 1, then thevalues of p', q' and r' become, respectively, p'=0.1l4, q'=0.273 and•RESEARCHES CARRIED OUT IN 1954•

39,

r'=O.613. From these values, the expected numbers of the four types
may be obtained:
0'=98.452, A'=40.034, B'=107.210, A'B'=16.308.
When each of these expected values is compared to the corresponding observed value, it is found that the observed value ishigher for 0 and AB types and lower for A and B types thanthe expected value, and the difference tetween these values issignificant. Thus, in this sample, as well as in several other samples
previously examined, the double heterozygote AB and the recessiveo types have a higher selective value than the single dominant types
A and B, and this situation seems to be responsible for maintaining
the polymorphism in a stable state.
Reference: KOMAI, T. 1953. Composition of wild populations in thelycaenid butterfly Neozephyrus taxi/a. Am Nat. 87: 87-95.

c) The land-snail Bradybaena. More population samples
were obtained from various localities in the country; thus the numberof samples of natural populations of this polymorphic snail has beenraised to 114. All these samples, except three only, contain the fourtypes, 0 (yellow unbanded), A (yellow banded), B (brown unbanded)
and AB (brown banded) in the proportion expected from the postulatethat these types are determined by a set of triple-allelic genes, with nodifference in selective value among them. In all the three exceptional
samples, the observed proportions of AB and 0 types significantly exceed the corresponding expected proportions, while the reverse in thecase with the A and B types,
Population samples were obtained by Prof. EMURA of Niigata
University from several localities in Niigata, sometimes with a nearly
20-year interval. A few of these samples show a slight but significant
temporal change in composition which took place during this interval.
This change seems to be largely due to migration caused by a changeof the topographic condition of the habitat which occurred during
this time.
Reference: KOMAI, T. and EMURA, S. 1955. Genetic studies of the polymorphic land snail Bradybaena simi/aris. Evolution 9: (in press).

40:

RESEARCHES CARRIED OUT IN 1954

25. Population Genetics on the Balanced Polymorphism inDrosophila rufa
(by Toshifumi TAIRA)

The female of D. rufa in the D. melanogaster group is distinctly
dimorphic. The" dark" type has a dark band on each abdominal
segment, much like that in the male melanogaster. The" light"
type has no such band, so that this type resembles the female
melanogaster. The male is monoformic.
Such a polymorphism is rare in the Drosophilidae; so far it is
known only in D. montium D 2) and in D. polymorpha3 ) besides thepresent species.
Two natural populations, one in a village in Kochi Prefecture andthe other in Asakawa near Tokyo, have been examined monthly byusing numerous traps. When the natural population is of the
maximum size, the relative adaptive value of different genotypes has
been found to be D/d> D/D > did.
On the other hand, in laboratory populations started at various
initial proportions of each genotype, the relationship of the adaptive
value in the equilibrium state is D/d > did > DID.
The difference between the two populations seems to depend on
the relative competitive ability in the larval stages of the heterozygote
with the two homozygotes. In all cases, the heterozogote has someselective advantage over either homozygote. Such heterosis seems to
be mainly responsible for the maintenance of balanced polymorphism.
1) WHEELER, M. R. 1949. Evolution 3: 268.
2) FREIRE-MAlA, N.
1949. Ibid. 3: 98.
3) DA CUNHA. A. B. 1949. Ibid. 3: 239; Nature (London)
171: 887 (1953).

G. POPULATION GENETICS OF RICE AND BARLEY

26. Analysis of Genes for Hybrz'd Sterility in the Varieties ofCultz"vated Rice(by Hiko-Ichi OKA)

The sterility in hybrids between distant varieties of rice has beena problem of wide interest among workers on this plant, becausesuch a study seems to afford them a key to classification of thevarieties. The hybrid sterility in rice is characterized by the following

RESEARCHES CXRRIED OUT IN 1954

41

features: (1) Crossing between distant varieties is as easy as between
nearly related ones; (2) The F I plants show no disturbance inchromosome pairing; (3) Micro- and macro-spores begin to deteriorate at a definite stage of development after the meiosis; (4) The
degree of sterility, if measured by the percentage of good pollen, is
not affected much by environmental conditions, and depends onthe combination of parental varieties. The degree ranges fromperfect fertility to nearly complete sterility. (5) The percentage ofgood pollen is generally similar to the percentage of seed setting;and (6) There is no significant difference between reciprocal crosses.
The F 2 of a semi-sterile hybrid generally shows a continuous variation in fertility. But when two related varieties, A and B, differingin fertility in the hybrid with C, are crossed in the manner (AxB) xC,
there appears a discontinous variation, possibly because of the relative
simplicity of genic difference between A and B. A few breeding
examples are presented in the table.
Segregation of fertile and semi-sterile plants in some crosses of(AxB)xC type.
~======~=======~-=-=

Cross% of good pollen

95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15

Num. ofplants563 x (219 x 221)
Homozygote*
Heterozygote*(108 x 143) x 553
563 x (l08 x 143)

3 3 3

(9)
1 2 2 1(6)

1 2 2 1(6)
1
2 3 4 1

15
17

(11)

1 3 8 8 6 7 4 3 8 9 5 16 12 9 5
(37)
(25)
(42)
2 1 3 12 4 3 1 4 5 2 2
(9)
(7)
(14)

104
30

* for the glutinous gene

Based on this finding, genic analysis was conducted on the hybrid
sterility. It has been revealed that the deterioration of gametes inhybrids is due to duplicate genes which act in the gamete as"development maintainers" (Gametic-Development, CD, genes). The
segregatipn of fertile and semi-sterile plants, as well as of homoand hetero-zygotes for the glutinous -gene, in 563 x (219 x 221) shownin the above table, can be understood by assuming that the varieties
219, 221 and 563 are of such constitutions as " +-X1X 2 ", " gl-X 1X 2 "42

RESEARCNES CARRIED OUT IN 1954

and" gl-XIX2" respectively, where Xl and X 2 are a set of duplicate
genes for gametic development, the double recessive combination ofwhich causes the deterioration of gametes carrying it. In this case,
Xl is linked with the glutinous gene gl. This linkage has been
deduced from the fact that, as seen in the table, fertile plants weremore numerous among the homozygotes for the glutinous gene, whilesemi-sterile plants were more numerous among the heterozygotes.
Furthermore, as is inferrable from this linkage, glutinous pollen
grains were much more numerocs than non-glutinocs ones in thesemi-sterile heterozygotes.
The second cross shown in the above table can also be fully understood by assuming two sets of GD genes. In this case, however, it
is seen that more semi-sterile plants were produced from 563 x (l08 x143) than from (108 x 143) x 563. This behavior suggests that thepollen with a dominant combination of GD genes has lower fertilizing
power, as the result of certation in favor of XIX:! or X I X 2•
For testing the validity of these assumptions, the F2 plants of" A xB" were crossed witn "C ", and in each progeny line the variation in good pollen percentage was examined. Moreover, the genotyt:es of the F2 plants and their frequences were expected under theassumption of genes based on the "(A x B) xC" experiment. First,
the probability that a given line carries the respective genotype wasestimated from the data for each line. These values were summed
up according to genotypes, and the sums of probabilities werecompared with the expected frequencies of the genotypes. Next, thevariation in fertility among all of these plants was compared withthe expected frequency distribution of fertility, which was obtainedby assuming that feltility variation due to various causes other than
GD genes follows the normal distribution. The recombination values
thus obtained from different sources were compared with each other.
These tests have proved that all the data obtained from theseexperiments conform well with the hypothesis of GD genes.

27. Variation in Fertility due to Gametic-Development Genes(by Hiko-IeW OKA)

As mentioned in the former article, there are only one or two
sets of "Gametic.Development" GD genes controlling the inheritanceof sterility in some crosses of "(A x B) xC" type. But occasionally,

RESEARCHES CARRIET Ot:T IN 1954,43

the outcome of such a cross shows a discontinuous variation withseveral peaks of frequency in the percentage of good pollen. In F2
also, similar discontinous distribution may be found, though in mostcases the variation appears to be continous. In these cases, the peaks
are found at definite points of the frequency distribution, namely,
75-70%, 60-55%, 45-40% and so on, and the mode of distributionagrees well with that expected under the assumption of several setsof CD genes. This fact seems to indicate that the CD genes
controlling fertility in a hybrid are rather numerous, and behave likepolygenes.
Thus, the continuous variations usually seen in the F 2 and later
generations may be explained by assuming certain sets of CD genes,
though other genes modifying fertility may also exist. A biometricgenetical study is then needed for analyzing the variations in fertility.
As an approach to such a study, the variation in fertility due to CD
genes has been theoretically investigated.
In the case in which a set of CD genes comes into consideration,
let the fertility in F 1 be r (=0.75), the frequency of semi-sterile plants
in F 2 be P(= 9~3S ' where I-s stands for the fertilizing capacityof double dominant pollen), and the propagation rate of semi-sterile
plants be I-s', the frequency of semi·sterile plants in F n will beI-s'p(pb)n-2, where b stands for I-ps"

The frequency of fertile plants

in F n will then be I-p(pb)n-2.
Considering k sets of CD genes, the frequency of fertile plants in
F,. will be {l_pcpb)n-2}k. Then the mean fertility (MFn) andvariance (VFn) may be written as follows:
MFn={1-(I-r)p(pb))n-2}kV Fn={I-(1-r-)pcpb)n-2}k-(MFn )2
The computations based on tl:ese formulas of the frequencies offertile plants, mean fertilities and variances in F 2 and later generations, have revealed that the hybrid sterility due to CD genes
vanishes rapidly with inbreeding generations and may disappear
almost entirely by F 4•
Analysis of variations in fertility with some actual data is still
under way. The results so far obtained suggests that the variationsin the progeny of semi·sterile hyprids are partly due the CD genes.

RESEARCHES CARRIED OUT IN 1954

44

28. Change of SegregaUon Ratio due to Gametze Development
Genes(by Hiko-Ichi OKA)

When a set of GD genes, Xl and Xz, operate in a hybrid, thesegregation ratios of the genes linked with this Xl or X z will be
modified more or less. For instance, in a variety cross "A-x1XZ " x"a-X1xz" (repulsion of A and X), if the recombination value between
A and Xl or a and Xl is p, the ratio of the gametes A and a will
become 1 +P : 2- p, and the ratio of AA: Aa : aa in F2 will be changed
as shown in the table below. (1-s stands for the fertilizing capacityof double dominant pollen XIXZ .)Table showing the modified Fz ratio Of AA: Aa : aa by the presenceof a set Of CD genes.
Repulsion:
Coupling:

AA
aa

Aa
Aa

aa
AA

Fertile (1+3p2)-sP~1+PJ (Z+6p-6p2)-s(1+Zp-ZP2) (4-6p+ 3P2)-s,Z-p,(1-P)
Semi-sterile
ZP(I-p)Z(l-Zp+ZPZ)
ZP(l-p)
Total(1+P)Z-sP(1+p) Z(1+P)(Z-p)-s(1+2P-2p2) (Z-p)2 -s(l- p)(2 - p)

In this table it is seen that the F 2 ratio may change from 1: 2 : 1
to 1: 4 : 4 (complete linkage, s=O), and the semi-sterile plants tend
to be heterozygous for A: a. Then, the phenotypically deminant class,in which semi-sterile plants are included, will hwe a lower meanfertility value and larger fertility variance than the recessive class.
If the frequencie3 of semi-sterile plants among plants with dominant
and recessive phenotypes are XD and XR respectively, the differences
between the two plant classes in mean fertility and fertility variancewill become as follows:
Mean fertility difference-d(xD-xR)
Fertility variance differenced2(xD-xR)(l-xD-xR)(d=0.25; the difference between fertile and semi-sterile plants in fertility). By use of these formulas, the values of XD and XR, andaccordingly the recombination value of A and x, can be found from
actual data.
The observed Fz segregation ratios of various monogenic characters have been found to divert in the direction and to the extent
theoretically expected. An example dealing with the apiculus coloration due to gene C is shown below.

RESEARCHES CARRIED OUT IN 1954

45

The variatian in fertility among colored and colorless F2 plants.
Cross(% of good Color at% of seed setting
No. of EXP'M
pollen in FI)
apiculus 1009080706050403020100 plants No.
ean
108 x 318(75%)
1 x325(60%)++

4 5 9 818 610
87833 261213 92121
91817 6 6 3

7
3
8
2

3 21
2
4
1

73 818 0.576
37 27.2 0.660
94 1170 0.485
62 39.0 0.520

Variance
0.05440.04700.03560.0275In these cases, by assuming one or two pairs of CD genes linked
with C: c, the values of XD and XR can be estimated from the data.
The frequency distributions for fertility computed from these values
agreed well with the actual data of variations observed in both coloredand colorless plant groups.
It has been noticed that in many crosses the glutinous gene, as
well as the apiculus color gene (linked with the former), has a generaltendency to decrease in frequency, while the phenol-reaction gene isrelatively stable, when these are judged by their segregation ratio in
F 2 • For the glutinous gene, the tendency was found that the lower
the F 1 fertility, the larger was the deviation of F 2 ratio from the
1: 2 : 1 ratio.

29. Change of Cene Frequency in Hybrid Populations of Rice(by Hiko-Ichi OK A)

Change of gene frequency is often seen in hybrid populations ofrice, when they are propagated without any artificial selection. Thismay be due to various causes such as:
Gametic-Gevelopment (CD) genes: As mentioned in the previous article, if a pair of genes for a character, A: a, are tightly linked
with a pair of CD genes, the ratio of AA: Aa : aa in F2 wil be 1:
4-s: 4-2s, where l-s stands for the fertilizing power of pollen
having a double dominant combination of CD genes. Of these gena·
2-stYJ:es, AA, aa and about a half or 4-s of the Aa plants will be
fertile, while the remaining half of the Aa plants will be semi-sterile.
Let the frequencies of semi-sterile Aa plants, fertile Aa, AA and aa
plants in F 2 be P,qhq2 and q3 respectively (P+Ql+Q2+q3=1), and the
propagation rate of the semi-sterile plants be u; the frequencies ofAA, .Aa and aa plants in F n will be given by the following formulas:

46

REsEARCHES CARRIED OUT IN 1954

AA:( 1 )n- q1B/ 1-P(1-u)A
21 q1A+qz(1+ PuA)-""2
1

Aa: ( 21 )n-Zql(1+2puB)+p(pu)n-Z / 1-P(1-u)A~ Q1A+ q3 <l+PUA)-( ; r-IQ1B !l-P(l-u)Aaa:(

A= l-(pu)n-z . B= 1-(2pu)n-Z )
I-pu'
1-2pu

2
2
4
1
When s=O, we have p= 9' Ql= 9' Qz= 9 and Q3= -9-' and111
when s=l, we have p= 3' Ql= 6' Q2= 3

1and Qr==: 6(el· Tableon p. 44). Putting these values into the above formulas, it is found thatthe ratio of AA: aa in F... is 2.5: 1 (s=O) or 1.67: 1 (5=1). The change
will take place in a relatively few generations, because of the rapid
elimination of the sterility due to CD genes.
However, such CD genes operating in the cross seem to be numerous in many cases. We should then consider a case where several
CD gene sets are linked with A or a. In such cases, the exact computation is rather difficult. The following formulas, however, can be obtained under the assumption of the absence of recombination between
those CD genes and 5=1. Let the number of CD genes (or thenumber of chromosomes which produce sterility if recombined withA: a) be m, and the average recombination value between A or aand the CD genes be p, the frequency of AA or aa plants in F n will be__!_) ± 3kP {l_(~)n-l}
( _:L
2
2"
3-P
3
'
2m - lwhere P=1-2P and k= -4(2 m +1)'

Difference in propagation rate: WhenAA,Aaandaaplants
have propagation rates 1, 1-h5 and 1-5 respectively, their relative
frequencies in F n will be as follows:)n-l1(-1-hs-AA:22(1+hs)aa:

l-hS' n-I(l-s)n-l- ( - - )22(1+hs-2s)Computation according to these formulas shows that the frequency
of AA or aa plants is determined largely by the value of s, and the

RESEARCHES CARRIED OUT IN 1954

47

effect of the value of hs is relatively slight. When hs=O, the frequencyof aa plants in F" may be written as
2,.-I(1-S)"-1_1
2" 1(1-2s)+2" 1(1-s)"-1_2s
In addition to the causes discussed above, there may be some othercauses which would affect the genotypic make-up of hybrid populations,such as the effects of sterility genes other than the CD genes, certation and competition. These will be dealt with at some other time.
The frequency of plants having the recessive phenotype due to thegene for apiculus coloration, phenol reaction, or glutinous endosperm
was examined with hybrid populations of F 2 to F 7 of several crosses
between distant varieties. The data obtained were ap..alysed by using
the above formulas, and the values of parameters such as k for the
CD genes and s for propagation rate were estimated. The frequencies
of a gene in different generations calculated from those values agreed
well with the actual data.
30.

Restriction of Cene Recombinatz"on in Hybrid Populatz"ons of
Rice(by Hiko·lchi OKA)

When two independent pairs of genes, A: a and B: b, are linked
respectively with two pairs of CD genes of the same set (Xl: Xl andX2 : X 2), gemetes possessing a recombination of the former genes in
the hybrid, such as AB in "A-Xl b-X2" X " a-Xl B-X2", will beeliminated in every generation, since they carry CD genes in double
recessive combination. In addition to this, the lowering of fertilizing
capacity of the Xl X 2 pollen will reduce another recombination, abo
In this caEe, the F 2 phenotypic ratio will be 2AB: 3-s Ab : 3-s aB :1-s ab, where l-s stands for the fertilizing capacity of the X I X 2pollen. Thus, the segregation ratio may appear as if it were due tothe linkage of A and B.
Let the recombination values between A-Xl and B-X2 be p and q
respectively, the frequencies of AB, Ab, aB and ab in FI gametic series
and in F 2 phenotype will be as follows:

48

RESEARCHES CARRIED OUT IN 1954

Frequency in
Fz gamete
ABAb

Fz phenotype (when I-s=0.5)

1-3(P+q-pq)+

1
15(4+6P+6q-4pq-pZ-qZ-pZq-pqz+3fil.qZ)(l-q+pq)-ol5-(5-P-6q+4pq+qZ+pzq+pqz-spzqZ)aB+(1-p+pq)-l5 (5-6p-q+4pq+PZ+PZq+PqZ-3pZqZ)ab-~-(I-pq)

11 (l+P+q-4pq-PZq-pqz+3pZqZ)
5

The genes for apiculus coloration (C: c) and for phenol-reaction(Ph: ph) in rice are independent of each other. They showed, however, in the F z of a semi-sterile hybrid, 414 x 325, a segregation ratio
as given below; repulsion between these two genes will be suggested.
Apiculus:
Ph.-reaction:

Colored+Observed Number:
96
49
Exp. Num. (9: 3: 3: 1):
127.8 42.5
(P=O. 086, q=O. 305): 92.8 49. S

Colorless

+

Total

69
42.5

13
14.2

X2=25. 54 (p<O.Ol)

65.1

19.5

Xb 2.94 Cp>0.20)

227

Since both of the genes C: c and Ph: ph have been found to beaffected by CD genes, the values of p and q may be computed by the
maximum likelihood method, using the above formulas, assuming theF 1 genotype to be~-=-~- ~~:.

The recombination values p=

0.086 and q=0.305 were thus found. The expected numbers based onthese recombination values showed good agreement with the observed
numbers, as shown in the above table.
This experiment may serve as an example to justify the theory
that genes linked with the CD genes of the same set behave as if
they were linked.
31. Do Chromosomes in TetraploM HybrMs between Dzstantly
Related Varz"e#es of Cultz"vated Rice Tend to Pair Selectz"vely?
(by Hiko-Ichi aKA)

In tetraploid F 1 hybrids between distantly related varieties ofcultivated rice, the fertility often becomes higher, and quadrivalentchromosomes become somewhat rarer, than in the parental tetraploidvarieties (eUA, L. D. 1951: Proc. Jap. Acad. 27 (1), OKA, H. 1.1954: Jap.

RESEARCHES CARRIED OUT IN 1954

49

Jour. Genet. 29 (3)). Thus the tetraploid hybrid behaves like anamphidiploid, and this behavior suggests the occurrence of selective
pairing of chromosomes derived from the same parent. However, as
far as the writer's experience with such cases goes, no significant
correlation is found between fertility and the number of quadrivalentchromosomes. Furthermore, it has been found that the fertility at
the tetraploid level varies markedly according to the diploid prototype,and it tends to be high in the plants showing vigorous growth, while
the tetraploid F 1 plants are generally more vigorous than their parents.
These facts seem to suggest that the improvement in fertility in F1
is mainly due to hybrid vigor.
The experiments reported here were conducted in order to evaluate
the relative probability of these two alternative hypotheses. First,
with two tetraploid hybrids showing high fertility in FI, a pedigree
breeding experiment was continued up to the F 7 , by selecting in every
generation plants with high fertility. It was found in this experimentthat in every generation segregation of plants with different fertilities
occurred and the mean fertility tended to go down gradually withthe repetition of inbreeding generations, and that high fertility was
always correlated with vigorous growth.
Also the F2 segregation ratios for several monogenic characters(phenol-reaction, apiculus coloration, seed coat coloration and glutinous
endosperm) were obEerved, by use of several tetraploid hybrids between distant varieties. All the segregation ratios thus found were
between the 35: 1 (random chromosome segregation) and 20.8 : 1(random chromatid segregation) ratios to be expected in an autotetraploid hybrid.
In F 2 plants of a cross in respect to glutinous endosperm, the number of glutinous homozygotes (gl gl gl gl) was significantly smaller
than the expected number. However, the F1 gametic ratio of + + :+ gl : glgl, computed from the F2 genotypic ratio, was as follows:
Observed
Frequency

+ +
+ gl
gl gl

0.328
0.551
0.121

Expected Frequency
Chromosome segregation Chromatid segregation
0.167
0.666
0.167

0.214
0.572
0.214

This gametic series shows that heterozygous gametes (+ gl),which are to be increased by selective pairing, were not more than

50,

RESEARCHES CARRIED OUT IN 1954

the ex'pl:!cted frequency based oli the assumption of random pairing.
The excess in ++ and the deficiency in glgl' were found to be due
to a certation in favor of non-glutinous pollen, in the same manneras in diploid hybrids.
TheSe experimental results contradict the hypothesis that tetraploidhybrids between distantly related varieties of cultivated rice behave
like an amphidiploid.

.
32. Genetic Analysis of Differentiation of Local Strains in theBarley" Hosogara No.2"(by Kanji GOTOH)a) Statistical differences among local strains.
"Hosogara No.2" is an old commercial six-rowed barley variety.
As was noted in a preliminary report (Ann. Rep. No.4, p.31), thisvariety includes some distinct local strains in respect to growth habit
and some other characters.
In the present study, population samples of six local strains were
statistically examined for several agronomic characters. In order toanalyze the interaction between the strains and local environments,
contrasting experiments were concurrently carried out at the Kitami
Branch of Hokkaido Agr. Exp. Stat., Kitami, and at the National Institute of Genetics, Misima.
The results of comparative experiments conducted in the years 1952
and 1953, have demonstrated some statistically significant differencesamong the strains, and that the greater part of these differences aregoverned by genetic factors.
Based on the degree of phenotypic similarities and dissimilarities,
the six strains were classified into three groups X(A, C); YCD); andZCE, F, G). It has been confirmed that the decline in vigor of plants
raised from grains produced at Misima affects the grain yield. Furthermore, the interaction between strains and local conditions is highly
significant relative to grain size. Among the four strains, C, D, E and
G, C showed the greatest change in grain size in response to thedifference in environment, while D showed the slightest change. The
range of variation in the date of heading in A strain coming from
Kitami, was 22 days in both years at Misima, and the parent-offspringcorrelation for this character was very high (0.763**). This wide
range of variation in the date of heading within the A strain seems
to be governed by polygenes. Polygenic variation was also foundR.E3EAR.CHE3 CARRIED OUT IN 1954.

51

within A and D strains for other characters, such as ear length andear density.
In F 1 hybrids between the three strains, C, E and G, heterosis wasobserved. The F 1 hybrid between G and C showed great vigor. In
respect to various performances, the differences between the northern
CA, C and D) and southern (E, F and G) strains were fairly wide.
This suggests a correlation between local conditions and strain formation. It is also highly probable that the original population of "Hosogara No.2." included an abundance of genetic variability.
Reference: GOTOH, K. Genetic analysis of varietal differentiation in cereals.
1. Statistical differences found among local strains of a barley variety .. Hooo-

gara No.2." Jap. Jour. Genet., (in press).

b) Difference in growth habits among local strains.
In cultivated plants, the date of heading, photoperiodic response andgrowth habit seem to be closely connected with strain formation, since
they are the characters which seem to have been subjected to natural
selection under the local environment. Experiments were designed todetect the differences in growth habit among the local strains.
The spring seeding experiments were carried out in 1952 and 1953
on the experimental grounds of the Institute. The samples of each
local strain were also grown in the air-conditioned greenhouse maintained at 20°C under a whole day illumination.
The greater part of the plants of the X group originating in Hokkaido showed a fairly pronounced winter habit. The A-S strain of
the same group, however, proved to be of a pure spring-habit type.
The D strain was of moderate winter habit, and the Z group originating in southern localities was homogeneous for its spring habit. Mostof the X and Y group plants and a few plants of the A-S strain andof the 2 group had long auricles on the first leaves. Most of the X
group plants and none of the 2 group plants had hairs on the leaf
sheath. In the D strain, the development of hair was strongly affectedby environmental conditions. The gene for short basal bristle (rachillahair) occurred mainly in the X group, though in low frequency.
In the air-conditioned greenhouse under the whole-day illumination,
all plants of A-S and 2 types produced ears 33 days after seeding on
the average, and the number of leaves on the stem was 6 in all plants.
With regard to this response, both types behaved similarly. However,some differences were found in the breadth of the leaves between the
A-S and 2 types, and in the presence (A-S) or absence (2) of hairs

52

RESEARCHES CARRIED OUT IN 1954

on the leaf sheath.
On the whole, my observations apparently have confirmed that differentiation of the various growth habit types among this variety iscorrelated with adaptiveness of the genotypes to the various local
environmental conditions, and these types were developed under the
influence of natural selection.
Reference: GOTOH, K. Genetic analysis of varietal differentiation in cereals.
II. Various growth habits in local strains of the barley variety" Hosogara No.
2." Jap. Jour. Genet., (in press).

c) Competitive ability of local strains. Experiments were
designed to examine the relation between the competitive abilityof the growth habit types and their differentiation. The results of theexperiments conducted in 1952 and 1953 with the A, A-S, C, D, E andG strains are as follows:
The competitive ability of each local strain was tested by comparing the relative respose of the strains to competitive conditions withthree tester varieties in each test. Under competitive conditions, tr.e
plants of the tested strains and the plants of the tester varieties weregrown alternatively in rows. Three barley varieties, Aizu No.7, Shinsakigake and Akashinriki were used as testers in 1952 and Aizu No.
7, Shizuoka-shiro-rokkaku No. 1 and Suihu in 1953. By previous experiments by SAKAI and GOTOH (1952, Ann. Rep. No.3, p. 58), therelative competitive abilities of the tester varieties were determined.
Based on the results of these two years, it was estimated that thecompetitive ability of "Hosogara No.2" is fairly high, although theevaluation was slightly different between the two years, and a considerable variation in this character was found among the local strains
in 1953.
E and G strains of the Z group show a higher competitive ability
than those of the other strains when tested at Misima. According to
the analysis of variance of the data in 1953, the term of interaction
between testers and strains regarding grain yield was statisticallysignificant at the 1 per-cent level. The order of the competitive abilityof the strains was

E> C> D> A-S.
These results suggest that the differentiation of the Z group fromthe original "Hosogara No.2" has been accelerated by its high com·
petitive ability in the southern localities in Japan.

RESEARCHES CARRIED OUT IN

II

1954

53

To examine this point more accurately, the following experiment
was started in 1952. Equal numbers of seeds of each strain of the Zgroup and those of C were mixed (e.g. C+E, C+G, etc.), and thesemixture3 were grown at Misima and at Kitami.
The constitution of these mixtures showed some changes when
examined after one year, and the proportions of the Z type in each
mixture tended to increase at Misima and to decrease at Kitami,
though to a slight extent.
The D strain, which showed the highest yielding capacity among
the examined strains in both years, had rather poor competitive ability.
Reference: GOTOH, K. Genetic analysis of varietal differentiation in cereals.
III. Competitive ability of local strains in the barley variety .. H030gara No.2."
Jap. Jour. Genet., (in press).

H. STUDIES ON COMPETITION
33. Effect of the Number of Competing and Non-competing Individuals on Competitional Increment(by Kan-Ichi SAKAI)

Two experiments were conducted to investigate the effect of different
numbers of competing plants on a plant placed in the center. In thefirst experiment, two varieties of barley, SZ and CK, differing in competitive ability, were used. In the second experiment, two upland rice
varieties, one of the" Japonica" type and the other, "Red Rice" ofthe "Indica" type, were used. The" Red Rice" is often found in thefield among the ordinary Japonica-type plants as a weed. In both ofthese two experiments, one variety was planted individually and surrounded by, (a) six plants of the same variety, (b) five of the sameand one of the other. (c) four of the same and two of the other, andso on, till all six plants were of the other variety. The distance
between any two plants in the combinations was fixed at 11.5 em.
In the first experiment, the competing plants among the six surrounders were grouped together to form a "competing area" aroundthe center, while in the second, the arrangement of competing andnon-competing pla~ts was at random.
The analysis of variance of the data showed that in the experiments,
the effect of the number of surrounding competitors was significant
and could be shown by a rectilinear regre3sion line. It was then concluded that the competitional increment or decrement of a plant would

54

RESEARCHES CARRIED OUT IN 1954

be proportional to the number of competing ones surrounding it, when
the inter-plant spacing is constant.

34. Polygenic Analysis of Quantitative Characters under Influenceof Intergenotypic Competition(by Kan-Ichi SAKAI and Hiko-Ichi OKA)

The theory of polygenic inheritance so far developed by Mather has
not taken into consideration the effect of intergenotypic competition.
However, in some characters of plants such as culm number, the effectof competition between different genotypes is known to be so largethat it should not be ignored in analyzing variance components in hybrid populations. It has been theoretically demonstrated, on the groundof a few premises, that the competitional variance in hybrid populations can be partitioned into the following four components:
C': Variance due to competitional increments between dominant
and recessive homozygotes for genes causing competition (c),G': Variance due to competitional increrr.ents between hetero- andhomozygotes (g),M': Covariance between c and d (Additive genetical increment),
N': Covariance between g and h (Non-additive genetical increment).
By using these components, formulas were constructed for F2 andother generations and for back-cross generations of autogamous plants
as follows:

Jc-D+~H+(C'+M')+~(G'+N')+El
2
4
2~3_D +~3 H +~-(C'+M')+ ~-(G'+N')+El
4

16

2

8

~D
+~H
+~(C'+M')+~(GI+NI)+El
4
8
2
4

~D+

:4 + ~

1

1

H(C'+M') +1;2(G'+N')+E1

1

gD +16H+-4(C'+M')+g(Gt+N')+~1

~D+~H
2
3

8
3
4-D + 32 H
77
gD+ 128H

+~M'

2
3
+TM'
7

+gM'

+~NI

8
3
+ 32N'
7 ,+ 128NRESEARCHES CARRIED OUT IN 1954

VB, + VB.:

_L
n +~H
+ (C'+M')+(G'+N')+2E1
2
2
D+ ~ H+2(C'+M') + ~ (G'+N')+2E2(Variances among line means are not given here, as they contain nocompetitional variance component.)

In some cases, C' and M' can be grouped into C" (Additivecompetitional variance), and G' and N' into Gil (Non-additive competitional variance). In some cases all of these competitional variancecomponents can be lumped together.
In order to test the fitness of these formulas to actual data, theinheritance of panicle number in two intervarietal crosses of rice
plant was analyzed. The results are given in Table 1.
Table 1. Showing the result oj experiments of" Pei-ku"
x "Taichung No. 65 ".
Methods

Estimated values of variance components
H
e"
Gil
El

D

Mather's
6.992
formulas ±l. 798
New
2.993
formulas ±2.013-3.344± 11.199-4.576±28.571

5.076
0.563
± 1. 181 ± 14. 638

16. 153±1. 276
13.474±0.714

E2

Deviationmean square±l. 473

1. 016

4.614

3.184
±1.114

1. 246

As shown in Table I, the mean square for deviations of the
expected values from the observed values is apparently smaller when
the formulas inclusive of competitional variances are used. The
heritability values for panicle number, computed from the variancecomponents, are also found to be comparable with the regression
coefficient of progeny line means on parental measurements when
the formulas inclusive of competitional variances are used. This is,however, not the case when the usual formulas of Mather are used,
as shown in Table 2.
Table 2. Comparing the heritability values according to Mather's
formulas and according to the new formulas.
Generation

Heritability value due toMather's formulas
New formulas
0.'247
0.330

0.080

0.336

0.098
0.103

0.382

0.105

Parent-progeny
regression

0.083
O. 124
0.144

56

RESEARCHES CARRIED OUT IN 1954

The same variance component analysis was conducted repeatedly
with another cross, "Taichung No. 65" x "O-chiam ", and similar
results were obtained at each time. Thus the heritability values
estimated from variance components can yield reasonable. values only
when the competitional variance is taken into consideration.
Heritability valueCompetitional variance
Considered
Not considered
0.138-0.1530.145

These experimental results seem to serve as a verification of thetheory of competition and formulas presented by the writers. They
also demonstrate that in biometric-genetical studies of quantitative
characters, the effect of competition should be considered, when thecharacter under discussion is affected by it.
35. How does a Mixed Population of Autogamous Plants Change
in Response to Intra-population Competition?
(by Kan-Ichi SAKAI and Hiko-Ichi OKA)

When different varieties of self-fertilized plants are mix-planted,
the relative frequency of these varieties will change with generations
as the result of differences in relative propagation rate and/or in competitive ability. Let us consider a mixed population of two varieties,
A and B, where A has a lower propagation rate, but higher competitive ability than B. We may denote the propagation rate of A as
x, taking that of B as unity. As to the competitional increment in
the propagation rate, it is assumed as a premise that an A plant will,in mixture with B, acquire a certain increment from each of the B
plants standing in proximity to it, while the B plant loses the sameamount. Since a definite design of planting is followed in an experiment, the number of plants surrounding a given plant can be expressedby a constant k. The number of B (or A) plants surrounding an A
(or B) plant wiIIthen be kb (or ka), where a and b stand for the
relative frequency of A and B plants, respectively, in the given
population. Then let the frequencies of A and B plants in the n-th
generation after mixing be an and bn, respectively; an and bn may
be written as follows:
an=an-l(x+kbn-lP)/Wn,

RESEARCHES CARRIED OUT IN 195457

bn=bn - 1(1-kan-lp)/Wn,
where
Wn=a n-l(x)+bn - 1 •In the same manner, let the initial proportion of A and B plants
be ao and bo respectively; the proportion in the n-th generation will
be related to the change in each generation as follows:
and

n-I

n

i-O
n-l

i-I
n

i-O

i-I

an=ao II (x+kbjp)/ II Wj,
bn=boII (l-kajp)/ II Wj.

andConsidering m varieties (A, B, ... M), the same computation leads us
to the following formula:

jn=in-l{Xj+an-l(Pa-Pj)k+bn - 1(Pb -Pj)k+ ···+mn-l(Pm-PJ)k}/Wn .
From this formula it is found that the population will reach anequilibrium when the relation pk=l-x (viz., competitive superiorityand propagation rate cancel e:lch other) holds good for every kindof genotype in the given population.
Data for a mixed population of two rice varieties (consecutive mixgrowing in Formosa, where the first and second crops alternate) wereanalyzed by using the above formulas. The below table shows theobserved and expected frequencies of "Taichung No. 65" mixed with
" O-chiam ".
The proportion of" Taichung No. 65" mixed with "O-chiam"in mix-harvested seed.

Year

Crop

Initial Proportion of Taichung No. 65
0.95
0.05
0.50
Obs. Exp.
Obs. Exp.
Obs. Exp.

1952

1st
2nd
1st
2nd

0.068
0.010
0.007
0.006

1953

0.048
0.009
0.008
0.001

0.510
0.206
O. 133
0.064

0.489
0.221
0.213
0.057

0.933
0.900
0.896
O. 778

0.948
0.877
0.872
0.719

In this calculatio:1, the relative propagation rates are denoted by
l-q and l+q in place of x and 1, and the values of pI (Pk; differencein competitive ability) and q in the first and second crops are estimatedseparately by the least square method. The values of pI and q obtained
are as follows:

RESEARCHE3 CARRIED OUT IN 1954

58

First crop
Second crop

l-q

P'

1. 056-0.155-0.511

0.716

These values indicate that the competitive ability as well as therelative propagation rate of Taichung No. 65 against O-chiam is much
lower in the second crop which grows under higher temperatures,
than in the first crop.
The expected frequencis in the above table have been obtained bysubstituting these values of pI and q into the formulas. They ~eemto agree well with the observed frequencies It may then be said
that the mixed population of self-fertilized plants cehaves according
to the rule given in these formulas.

36. Competz'tion Experiment with Diploz"d and Autotetraploid
Races of Rice(by Kan-Ichi SAKAI and Yasuo SUZUKI)

Four varieties of rice and their autotetraploid races were tested toinvestigate their competitive ability. All the varieties used, Sekitori,
Sensho, Sen-Ichi and Sin-Riki, were of the" Japonica" type. The splitplot design with four replications was u~ed throughout the experiment.
Planting was made individually in rows, the interval between the rows
being 30 em, and the interplant spacing 12 em. The comJ:etitive ability
is expressed by the difference between the mixed plot and pure standplot in quantitative characters such as plant weight, culm number,panicle number, panicle weight and seed weight, taken on an individual plant basis.
Analysis of variance of the data showed that the effect of competition was highly significant in all of these characters. As an example,
mean plant weights in the two chromosome races in pure stand andin mixture are given below.
Mean plant weight in diploid and autotetraploid races of jour varietiesof rice, each in pure stand and in the mixture with the otherchromosome race Of the same variety (in gnc).
Diploid

Variety
Sekitori
SenshoSen·Ichi
Sin-Riki

Pure
30.45
29.84
27.85
35.55

Mix
38.73
82.35
34.32
45.57

Autotetraploid
MIX

Pure
22.62
25.02
19.75
21.11

21.45
24.88
15.15
19.27

RESEARCHES CARRIED OUT IN 1954

59

The table shows that in all these four varieties the autotetraploidraces had lower competitive ability than their diploid progenitors. Thisagrees well with the result of a similar experiment with barley varieties
reported in the preceding issue of this Annual Report.

37. Competition Experiment with two Nicotiana Species and their
Allotetraploid.
(by Kan-Ichi SAKAI and Yasuo SUZUKI)

In order to estimate the competitive ability of allopolyploid plants,
an experiment was conducted with Nicotiana alata (n=9), N. plumbaginifolia (n=10) and the allotetraploid synthesized from the two
species, N. dzplumbalata (n=19). Planting was made with the plant
to be examined in the center, and six plants to compete with thecentral plant around the latter. The interspace between any twoplants was fixed at 24 em. The split-plot design was employed withthree replications. Plant height, capsule number and flower number
were measured on an individual plant basis. Analysis of variance ofthe data obtained showed that the effect of competition was highly
significant for plant weight and for flower number.
Mean values of plant weight, capsule number and flower numterin pure stand and in mixture are given in the following table.
Mean values of plant weight, capsule number and flower number
oj three Nicotiana species in pure-stand and in mixture
with one of the other two species.
A:

N. alata

P:

N. plumbaginijoliaAP:N. diplumbalata

Competing species
Characters
Plantweight(gm)

CapsulenumberMean valua
APAP
Com pet.
effectA
P
AP
Compet.
effect

A

P

AP

78.9
153.2
188.7

49.4
116.6
82.9

53.9
125.7
108.9

104.0

82.9

96.2

56.4
142.0
115.0
L.S.D. (5%)=
5.81

24.5
65.9
30.6

9.5
40.7
20.4

12.6
48.6
17.8

15.5
51. 7
22.9

40.3

23.5

26.3

60

RESEARCHES CARRIED OUT IN 1954

Flowernumber

A

P
AP

4.8
27.1
9.3

1.1
18.9
4.3

0.7
13.8
6.5

Compet.
effect

13.7

8.1

7.0

2.2
19.9
6. 7
L.S.D. (5%)=
3.60
-----

In the table it may be found that of the parental diploid species,
alata is a weak competitor and plumbagz"nifolia is much stronger,
while the synthesized dz"plumbalata behaved approximately the sameas, or a little stronger than, the stronger parental species. It is suggested that allopolyploidy has an evolutional significance which is dueto the superiority of such plants in competition.

1. RADIATION GENETICS OF WHEAT

38. Gene Mutattons in Einkorn Wheat induced by X-rays(by Seiji MATSUMURA and Taro FUJII)

In order to study the relation between the frequency of gene mutations and the dos3ge or wave length of X-rays, dormant seeds of Triticum monococcum were exposed to X-rays at 180 KVP, 3mA, without
a filter. The dosage ranged from 5,400 r to 13,500 r units. The results
are shown in Table 1. The frequency of head progeny with induced
mutations in the X2-generation increases with X-ray dosage in a
linear relation. There was some difference between the sensitivity ofthe varieties to X-rays.
Table 1. Relation between dosage or wave length oj X-rays andfrequency Of induced mutations in T. monococcum.
Voltage(KVP)% of head progeny in X2 with mutants in

Dosage(r)/

Control
180
180
180
130
80

5,400
8,100
13,5008,100
8,100

var. vulgare

var. flavescens

0(18
4(60
7/55
10/27
2/55
3(54

( 0.00)( 6.67)(12.73)(37.04)( 3.64)( 5.56)

0(14
5/44
5/26
6/15
4/43
4(46

"

( 0.00)(11.36)(19.23 )(40.00)( 9.3l)( 8.69)RESEARCHES CARRIED OUT IN 1954

61

At the same dosage (8,100 r) and target distance, but with varying
kilovoltage (80-180 KVP) and time of exposure, the mutation fre.
quency increased with the decrease of wave length, though not in a
linear relation (Tab. 1). In this case a very similar difference in sensiti.
vity between the two varieties has also been confirmed. These findingson the relationship between the mutation rate and X-ray quality arelargely in good accord with those in previous experiments which wereprimarily concerned with the frequency of chromosome aberrations.
These facts can be explained on the basis of the difference in the distribution of ionization within the nuclei and the chromosomes: in hard
X-ray irradiations, the majority of the resulting ionizations are more
scattered than when soft X-rays are used.
About 70% of the mutants were chlorophyll abnormalities, of which
about half were albina and the rest includes chlorina, virido-albina,basi.viridis, striata (white striped) and so on. Besides, there were:
early, irregular ear, slender (narrow leaf), dwarf, shrunk, etc. Thesemutants behaved as simple Mendelian recessives. Virescent mutants
with partly green leaves, virido-albina and basi-viridis, mostly diedout during the winter in the field, but not in the green-house. Moreover, most of these mutants recovered comp~etely and produced green
leaves under the light of a fluorescent lamp..
As to the nature of the mutants, the same types were often observed in both varieties, flavescens and vulgare, showing a pronounced
parallelism. But var. flavescens always showed a higher tolerance to
X-rays than var. vulgare.

39. Chlort'na Mutants t'n Et'nkorn Wheat ttzduced by X-t'rradt'att'on(by Taro FUJII)

Chlorina mutants appeared in the X2-generation from dormant seedsof Triticum monococcum L. var. flavescens KORN. treated in 1951 with
X·rays of 5,400 r dose, at 180 KVP, 3 rnA tube current, 13 cm target
distance, without filter. This type of mutation behaves as a simple
recessive.
Chromosome conjugation of the mutants is always normal, and 7u
are formed. Nevertheless, the mutants are somewhat inferior to thenormal in plant height and fertility; that is, the plant height is about
25 cm lower, the fertility is about 15% lower, and the time of heading
and ripening is about 10 days later as compared with the normal plants.
The difference in chlorophyll content between the normal and mu-

62

RESEARCHES CARRIED OUT IN '1954

tant plants was examined in the middle of May, 1954. The amountof chlorophyll was measured by extinction (E) at the wave length of4230 A with the BECKMAN-spectrophotometer. The amount of chlorophyll in the mutant was about half that in the normal plant. The
chlorophyll components -were separated into a and b by the paperchromatographic method. The amounts of chlorophyll a and b in
the mutants were also about half, as compared with those in the normal plant.
Next, the activity of cytochrome oxidase was measured with a WARBURG constant volume respirometer. The manometric rate 10, 30 and
60 minutes after treatment was 0.962, 2.743 and 5.214 p.1. respectively
in the normal plant, while it was 0.429, 1.413 and 2.609 p.1. respectively in the mutants. Thus, the manometric rates of the mutants wereabout half those of the normals. These results were in good accord
with those obtained in a previous experiment (1953).
The chlorophyll is presumably under the control of the activity ofcytochrome oxidase. The close relation between the chlorophyllamount and the activity of cytochrome oxidase awaits further investigation. The differences seen in morphological characters and in
viability between the normal and mutant plants are apparently due
in large measure to the reduction of the activity of cytochromeoxidase.
Reference: FUJII, T. 1955. Mutations in Einkorn wheat induced by Xrays. I. Chlorina mutants. Proc. Japan Acad. 31 (2): 88-92.

J. CYTOLOGY AND GENETICS OF NICOTIANA
40. Cytogenetic Studies on the Genus NtCotiana, VI.
(by YO TAKENAKA)a) Reduction divisions in hybrids between N. tabacumand two other species.
The three species, N. paniculata (n=12), N. tomentosz'formis (n=12) and N. alata (n=9), were crossed each with N. tabacum. The<:ross N. panzculata x N. tabacum and its reciprocal gave no seed,
while a few seeds were obtained from the cross N. tabacum x N. alata,
and abundant seeds were produced from the cross N. tabacum x N.
tomentosz'formis.
The hybrids obtained from the seeds of N. tabacum x N. alata wereRESEARCHES CARRIED OUT IN 1954'

63

polymorphic in external characters, in agreement with KOSTOFF'S description (1941-43). The plants from the cross Pindorama (a variety
of tobacco) x N.alata were generally intermediate with regard to theexternal characters of both parents, except for the length of stalk andleaf size which were diminutive, while the plants from Bright Yellow(another variety of tobacco)xN. alata resembled N. tabacum morethan N. alata, but the size of the organs was intermediate between
the two parents.
The external characters of the hybrid N. tabacum x N. tomentosiformis was in accord with the descriptions of KOSTOFF (1941-43) andmany other previous investigators.
The above two hybrids showed considerable irregularities in the
meiotic behaviour in the PMC's. Polysporous PMC's were often observed and the hybrid N. tabacumxN. alata was completely sterile,
but the hybrid N. tabacum xN. tomentosiformis gave some seeds.
F 1 N. tabacum (n=24)xN. alata (n= 9) showed 33 chromosomesin the root tip cells, the sum of the haploid chromosome numbers of
the parents.
At the first metaphase in the PMC's of the plants, 1-6 bivalents
were observed, and the most frequent configurations found were 311+
171, and 411+151, although rarely trivalents occurred. According to
KOSTOFF'S description (1941-43), the hybrids, N. tabacumxN. alata
and N. tabacum x N. Sanderae contained 5-9 bivalents and also somepolyvalents.
In a previous paper, the writer reported that the hybrid N. tabacum x N. sylvestrzs had 0-9 trivalents with the mode at 3. In the
hybrids N. tomentosaxN. sylvestrzs, GOODSPEED (1934) counted 0-7
bivalents with the mode at 2-3, and KOSTOFF (1941-43) found a similar
meiotic pairing behaviour. In the hybrid, closely related to the former
N. tomentosiformisxN. sylvestris, KOSTOFF (1941-33) observed somewhat fewer bivalents. These facts suggest that there are three pairsof homologous or partially homologous chromosomes between the tomentosa or tomentosiformis and the sylveslrzs genomes. Accordingly,
the bivalents found in the F 1 hybrid N. tabacum x N. alata cannot be
attributed simply to homologous relation between the two subgenomesof N. tabacum and the alata genome.
At first metaphase in the PMC's of FIN. tabacum x N. tomentosiformzs, the most frequent configuration found was 1211+121 and thenext, llu+1m+1l1. Besides these two main configurations, a few
deviations from them were found.

RESEARCHES CARRIED OUT IN 1954

64

. This meiotic behaviour agrees with GOODSPEED'S findings (1934) inthe same hybrid. Since the studies on the hybrid N. tabacum x N.
tomentosa by GOODSPEED and CLAUSEN (1928), N. tomentosa or N.
tomentosiformis is recognized by many tobacco investigators to be oneof the ancestors of N. tabacum.
b)

Reduction divisions in several hybrids between
NicoUana species.

i) N. sylvestris x N. otophora. Concerning this hybrid or its reciprocal, no report has been published, so far as I know. In external
morphology, F 1 sylvestri's x otophora is more similar to N. sylvestristhan to N. otophora, but the flower tube is markedly shorter thanthat of N. sylvestrzs.
At the first metaphase of the PMC's in F 1 sylvestrzsxotophora, 0-5
bivalents, mostly 2-3, were counted; also secondary associations between univalents were. frequently observed. At the first and thesecond anaphase, a few chromosome bridges were found. At the
tetrad stage, besides the four ordinary microspores, smaller microspores were occasionally detected. The hybrid was completely sterile.
The hybrids N. tomentosa x N. sylvestris and N. tomentosiformis x
N. sylvestris, closely related to the hybrid N. sylvestris x N. otophora,
have been studied cytologically, the former by KOSTOFF (1941-43) andalso by GOODSPEED (1934), the latter by KOSTOFF 0941-43). The results of their investigations are very similar to those of my study.
The number of pairing chromosomes, presumably reflecting the gradeof homology between the sylvestris and the tomentosa group genomes,
is about 2-3.
ii) N. alataxN. Sanderae. N.Sanderae is assumed to be a perpetually segregating hybrid between N. alata and N. Forgetiana. Its
flower colour varies between white, dark red and purple. Genetic
factors conditioning self-sterility are assumed to be the main cause
for maintaining high heterozygosis in the forms of N. Sanderae.
F 1 alata x Sanderae yielded various segregation forms.
The reduction division of the PMC's of F 1 atata x Sanderae proceeded quite regularly from the prophase to the tetrad stage, but,besides the chromosome configuration 9Il at the first metaphase, 8Il +21 was occasionally found. This phenomenon suggests that a small
portion of different chromatin or an inversion is present between thealata and Sanderae chromosome sets.

RESEARCHES CARRIED OUT IN 1954iii) N. rustica x N. paniculata. This hybrid has been very frequently produced, like the· hybrid N. tabacumxN. sylvestris, andinvestigated by a great number of biologists since the premendelian
period. The external characters of this hybrid agree with descriptionsof previous investigators.
At the first metaphase of the PMC's, 2-12 univalents, mostly 7-10
with the mode at 9, were counted. This range of the univalent number suggests the presence of many PMC's containing 2-5 trivalents
besides 7-10 bivalents, because polyvalents higher than trivalents seldom occurred. In fact, the typical configuration of the chromosome
complement was 911+3m+9r.
In the same hybrid, KOSTOFF and RADJABLY (1934) observed mostfrequently 1211+121 and sometimes lln+1nr+lll. Also GoODSPEED(1934) found very rarely 1011+ 2m+101, besides the configurations mentioned above.
There are some differences between my observations and thosemade by KOSTOFF and RADJABLY and also by GOODSPEED. KOSTOFF(1941-43) counted 1-5 bivalents in the hybrid N.paniculataxN. undulata, while GOODSPEED (1934) observed a wider range, reaching 7
bivalents in the same hybrid. These findings suggest the presence ofseveral semihomologous chromosomes between the paniculata and theundulata genomes. Accordingly, at the first metaphase of the PMC'sof the hybrid N. rustica x N. paniculata, several trivalents seem to benaturally present. The discrepancies between the results obtained byprevious investigators and myself in the cytological examinations of
N. rustica and N. paniculata may be due to different varieties ofthese species used in the cross.
c) Reduction divisions in N. Langsdorffii. The haploid
chromosome number of N. Langsdorffii is nine, as in N. alata,
N. Sanderae and N. bonariensis. AVERY (1938) reports that thisplant shows nine bivalents at the first metaphase of the PMC's;
however, I have most often observed the configuration 711+ 11V, occasionally 9Il, and rarely 7n+1m+1r at the diakinesis and also at thefirst metaphase. The quadrivalent is N-shaped at the first metaphase.
The two middle elements are the largest of all the chromosomes andthe two terminal ones are about the same in size as the other chromosomes. At the first anaphase, the four elements of the quadrivalent are distributed equally between the two poles.
At the meiosis of haploid N. Langsdorffii KOSTOFF (1930,1938,1941)

RESEARCHES CARRIED OUT IN19S4

observed one, two or three groups of two chromosomes in close proximity (secondary association) during the first metaphase, and occasionally one and rarely two bivalents during the first metaphase andearly anaphase. Accordingly, he assumed that the basic chromosome
number of the ancestral plant of the genus Nicotiana might have beensix. My observations also suggest that the basic chromosome numberof this genus is lower than 9.

41. MutaUon in Tabacco Plants Induced by X-rays(by Seiji MATSUMURA and Taro FUJII)In previous experiments (1951-1953), dormant seeds of Bright Yellow
and Dixie Bright 101 were irradiated with hard X-rays at 180 KVP,
3 mA, without filter. The dosage was 15,000-50,000 r. From the results
obtained, it has been surmised that the mutation rate is about 7% at
15,000 r, 20% at 30,000 rand 40% at 50,000 r, and also that the adequate
dosage of X-rays for radiogenetical study of induced mutations intobacco is 30,000 r. In 1954, dormant seeds of Dixie Bright 101 were
exposed to the same dosage (30,000 r) of soft and hard X-rays withvarying kilovoltage (20-180 KVP). The germination rate of treatedseeds increased generally with the increase of kilovoltage, and thegermination was more delayed and became more uneven at 20 and
50 KVP than at 180 KVP.
The mode of inheritance of many mutants obtained in the previous
experiments at 180 KVP was investigated in the (X3 - X5)-generationsof both the varieties. Many recessive mutants were observed, suchas early, pubescent, round, narrow, oblong, mottled, yellowish green,
yellow-spotted leaves, yellow petiole, dwarf, lethal, etc. Several kindsof mutants, for instance early, pubescent and narrow leaves, appeared
again by segregation even in the X3 - and X4-generations. In the
tobacco plant, relatively few chlorophyll mutants are found, comparedwith those in diploid wheat and barley. This may be due to the factthat the tobacco plant is tetraploid.
The "early" mutant of Bright Yellow appeared after 10,800 rirradiation at 90 KVP in one of the earliest experiments (1950). This
plant flowered about 2 weeks earlier, and was a little smaller than
the normal plants. Its leaves, as well as those of the "pubescent"mutant of Dixie Bright 101 derived from 30,000 r-irradiation at 180 KVP
in a previous experiment, seem to be of a good quality. Both mutants may prove to be useful for improvement of the tobacco breeds.

RE3EARCHE3 CARRIED OUT IN 1954

67

K. GENETICS, CYTOLOGY AND BIOCHEMISTRYOF SOME PHANAEROGAMS

42. HybridzzaUon Experiments with Citrullus vulgaris andCitrullus colocynthis(by Kazuo FURUSATO and Akira MIYAZAWA)

In the course of our breeding work with seedless water melons, across between a water melon (Citrullus vulgaris) and Colocynthe(C£trullus colocynthis) was attempted with the following results:
1) When Colocynthe pollen was used on water melon, the parthenocarpic fruits grew to normal size and ripened well. No germinating
seeds were produced. Most of the seeds remained immature, only a
few attaining normal size. Therefore, Colocynthe pollen could be used
for inducing parthenocarpy in water melon.
2) When water melon pollen was applied on Colocynthe, fruits ofnormal size were produced, and the seeds germinated well. Thus, thehybrid could be obtained only in this direction of the cross.
3) When Colocynthe (diploid) was crossed with tetraploid water
melon, germinating triploid seeds were produced in abundance, incontradistinction with our experience that diploid water melon does
not produce ripening or germinating seeds when tetraploid strains
furnish the pollen. Those triploid seeds germinated well, and grew
into triploid plants with seedless fruits with many Colocynthe characters: The pericarp was solid and preservable for a long time, but
it was too bitter for human consumption. If it were possible totransfer from this cross to diploid water melon the ability to produce
triploid progeny when pollinated by tetraploid strains, this would
mean a remarkable progress in our seedless water melon breeding for
two reasons: (i) It would become very easy to produce large amountsof triploid seeds, and (ii) the germination of the seeds so obtained would
be much better, since their seed coat would be diploid in contrast to
the triploid seeds obtained from pollinations of tetraploid plants withthe pollen of diploids, whose seed coat is tetraploid, requiring tedious
manipulations before sowing.
4) It is noteworthy that Colocynthe x water melon hybrids, both
diploid and triploid, are vigorous, prolific, fast growing and to a certain extent disease resistant. The striped pattern of the exocarp andthe texture of the endocarp of Colocynthe are dominant over thecorresponding characters of water melon.

68

RESEARCHES CARRIED OUT IN 1954

43. Karyotaxonomic studies in Poaceae, II(by Tuguo TATEOKA)

In addition to the species described in the last report, the species
listed below were examined cytologically during the current year.
These findings seem to allow some phylogenetic conjectures about thegenera Brachypodium and Brachyelytrum, and some cytotaxonomic
understanding with respect to the genera Digitaria, Setaria andCalamagrosits.
Species
Bromus secalinus
B. mollis
B. rigidus
B. remotiflorus
B. catharticus
B. tectorum
Agropyron semicostatum
A. Gmelini?*
Elymus dahuricus?*
E. verginicus
E. sibiricus
Lolium multiflorum
L. perenne
Brachypodium pinnatum
B. distachyon
Festuca myuros
F. arundinacea
F. elatior
F. extremiorientalis
Poa hayachinensis
P. Matsumurae
P. trivialis
P. sphondylodes
Glyceria depauperata
Torreyochloa natans

2n
28
28

56
14
42
14
28
28
42
28
42**

14
14
28
30
42
42
14
28
42
28***
28
28
20
14

Species2nAgrostis palustris
42
Calamagrostis Fauriei
28
C. longiseta
28
C. Pseudo·Phragmites
28
C. sachalinensis
56
C. purpurascens
28
Polypogon fugax
42
Alopecurus aequalis var. amurensis 14
Arrhenatherum elatius var. bulbosum 28
Trisetum bifidum
28
T. sibiricum
14
Brachyelytrum japonicum
22
Stipa scribneri
40
S. spartea
44
Achnatherum pekinense
24
Phaenosperma globosum
24
Pleioblastus Matsunoi
48
P. angustifolius
48
P. sp. (P. Simoni?)48
P. sp. (P. yoshidake?)48
P. sp. (P. communis?)48
Pseudosase japonica
48
Sinoarundinaria aurea
48
Sinobambusa tootsik
48
Sasamorpha mollis
48*) Plants collected in the Himalayas, the identification uncertain.
**) Plants collected in the Himalayas, their chromosome number is different
from that of other plants of the same species collected at Nagano-ken, Japan,
previously reported (2n = 28).
***) Plants collected on Mt. Mitsutoge in Yamanasi-ken, has a different chromosome number from that of the plant obtained at Mt. Yari in Nagano-ken
previously reported (2n=70)

69

RESEARCHE3 CARRIED OUT IN 1954

Semiarundinaria fastuosa
Arundinaria sp. (A. atamiana?)
Sasa sp. (S. nipponica ?)Cleistogenes Hackelii
Eragrostis megastachya
E. bulbillifera
E. multicaulis
E. pilosaE. poaeoides
Eleusina indica
Sporobolus japonicus
S. elongatus
Muhlenbergia japonica
M longistolon
M hakonensis
Zoysia japonica
Z. macrostachya
Cenchrus echinatus

48
48
48

40
20
40
40
40
40
18
40
36
42
42
40
40
40
68

C. paucijlorus

34

Setaria chondrachne
38
Digitaria chinensis
18
D. adscendens
54
D. violascens36
ca. 54
Oplismenus undulatifolius
Paspalum dilatatum
50
P. Thunbergii
40
Pennisetum alopecuroides
18
Cymbopogon torti/is var. Goeringii 20
Eccoilopus colutifer
40
Microstegium vimineum
var. polystachyum
40
Hemarthria japonica
18
Andropogon brevifolius
20
Pseudopogonatherum quadrinerve 40
Sorgum nitidum var. majus
20
Phacelurus latifolius
40

44. Effects of Extracts from Two Poisonous Plants upon Living
Plant Tzssue(by YO TAKENAKA)a) Extract from Glorz"osa superba. PARTHASARATHY (1941)
first reported that the alkaloid gloriosine extracted from Glorzosasuperba causes doubling of chromosomes. This finding was confirmed
more recently by KUMMER (1953).
The alkaloid was extracted from fresh root stalks of this plant by
the following method. Fresh root stalks (28,5 g) were macerated
with 42 cc water, centrifuged, and the supernatant fluid was sterilized(lOO°C, 4 min.) and filtered.
The effect of this filtrate upon plant tissue was examined in thefollowing way: (1) Tumor-test on growing root-tips soaked inaqueous solutions of the filtrate at various dilutions; (2) Dwarftumor test on seedlngs germinated from seeds soaked in the solutionof the same extract; (3) C-mitosis test on growing root-tips dippedin the same solution.
The tumor tests on growing roo-tips were repeate:l twice in thespring of 1954 with AIUum scordoprasum var. vivzparum and Vicia
faba. When the root-tips of AlUum were tre3.ted with this filtrate

70

RESEARCHES CARRIED OUT IN 1954

diluted to 1/2 concentration, they showed withering. When the concentration was made 1/4, tumors appeared, much as in the case oftreatment with 0.04-0.1% colchicine solution. The largest tumors
were formed by treatment with the filtrate diluted to one-eighth.
With 0.01% colchicine solution, ev€m small tumors are rarely formed.
In the experiment with root-tips of Vz'cia [aba, the material was
soaked in various concentrations of the filtrate. They showed withering in 1/4 concantration, grew small tumors in 1/8 concentration andlarge tumors in 1/16 concentration. After treatment with colchicine
solution of the concentrations, 0.1, 0.04 and 0.01, small tumors, large
tumors and insignificant tumors were obtained, respectively.
The dwarf-tumor tests in seedlings were carried out with a Raphanus species in the spring of 1954. The seeds of this species were
soaked in solutions of different concentrations of the filtrate for 72
hours. The proportion of dwarfed seedlings carrying tumors causedby this extract reached about 70%.
The root-tips of A1Uum scorodoprasum var. vz'vz'parum treated for6 hours with 1/4 cone. of the filtrate showed many cells with beautiful
figures of C-pair chromosomes. Materials treated for 6 hours with
1/2 cone. of the filtrate and washed afterward also showed doublingin many cells when observed 18 hours after the treatment.
b) Extract from Zephyranthes candz'da. ERDMAN and
EMMEL (1950) described Zephyranthes atamasco as a poisonous plant;
cases of poisoning in horses, cattle and chickens have been reported.
TYLOR, CARMICHAEL, McKENNA and BURLAGE (1951) studied Cooperz'apedunculata, a plant closely related to the former, and found thatextracts of this plant inhibited tumor growth.
The author examined the toxicity of Zephyranthes candz'da in thespring of 1954. The extract from the bulbs of this plant was madeby the following method. 26.2 grams of fresh bulbs were macerated
with 20 ee water + 13 cc alcohol, filtered by suction and concentrated
to IDee.
The effect of this extract upon plant tissues was examined in thefollowing way: (1) Growth-inhibition-test on root-tips soaked in thisextract; (2) Survival-test on growing root-tips and seeds soaked inthis extract; (3) C-mi.tosis test on growing root-tips dipped in thesame extract.
In the growth-inhibition test, growing root-tips together with bulbs
of Allz'um scorodoprasum var. vz'vz'parum were soaked in the extract

RESEARCHE3 CARRIED OUT IN '1~4

for 2, 4, 6, 8 or 18 hours. After 8 days, the roots of the control plants
were found to have grown about 3cm in length, whereas the roots
treated for 2 hours with this extract grew only about 1 em, and in
the remaining lots there was entirely no growth.
Survival-test on growing root-tips were carried out with the sameplant. Roots treated with the extract for 1 or 2 hours were soaked
in an aqueous soluion of neutral red after washing. In a few days,
the treated roots had selectively absorbed so much neutral red thatthe solution became nearly colorless.
Survival-tests on the germination of seeds were performed withseeds of a Raphanus species. Seeds soaked for 24 hours in the extractand in aqueous solutions of 0.5% cone. of two kinds of maleic hydrazide,
were sown in petri dishes after washing. The seeds treated bymaleic hydrazides showed slightly delayed germination, and thegrowth of seedlings was obviously inhibited. The rate of germination,.
however, was about the same as that in the control. The seeds
treated by the extract showed no sign of germination even after aweek; germination took place only when the seeds were plantedin the soil.
The seeds of the same species tre'3.ted with the extract for 1,2,3
or 4 hours were sown in petri dishes. They were delayed in germ;ntion as compared with the control; the degree of delay was nearly
proportional to the duration of the treatment, and the rate ofgermination decreased in inverse proportion to the duration.
In the C-mitosis test, growing root-tips of Allium scorodoprasum
var. viviparum treated with the same extract for 1, 2, 4, 8 or 24 hours
were observed microscopically. Materials treated for one hour showed
scarcely any difference from the cOLtrol. In materials treated for twohours, many nuclei and nucleoli had a swollen appearance, and a few
vacuoles were recognized in some nucleoli. Prophase chromosomes
were somewhat puffed up, and occasionally showed two sister strands.
Metaphase and anaphase chromosomes appeared shortened and plump.
In materials treated for four hours, these changes were much more
pronounced. Moreover, metaphase chromosomes showed signs of Cpair, and at the anaphase stage, various multipolar sp;ndles were often
observed. In the materials treated for 8 and 24 hours, all the changes
described above were much more pronounced, and the separation of
the centromeres in C-pair resembled those appearing after colchicine
treatment.

72

RESEARCHES CARRIED OUT IN 1954

45. Analysi's of Flavone Pigments in Triticum and RelatedPlants under Consideration of their Genome Constitution(by Toru ENDo)

The relation between genome constitution and flavone ingredientswas investigated by means of paper chromatography in eleven speciesof the subtribe Triticinae and one species of Hordeum. The plants
and their genome constitutions are as follows; Triticum vulgare andT. Spelta (AABBDD), T. Khapli, T. polonicum and T. dicoccum(AABB), T. monococcum (AA), Aegilops squarrosa (DDDD), Agropyrum glaucum (BBEEFF), A. elongatum (BBEEEEFFFF), Secale
cereale (RR), Haynaldia villosa (VV) and Hordeum vulgare (2n=14).
Methanol extracts of fresh le3.ves were analyzed by two-dimensional
paper chromatography. The solvents used were (1) mixture ofo-creso1jiso-propanol/water (5: 1: 4, v/v) and (2) 10% acetic acid.
Lead subacetate was found to be most effective as the color reagent.
After treatment with this color reagent, pigment spots in all chromatograms became either yellowish or brownish. The number of color
spots per chromatogram ranged from 6 to 11 ; in addition, a few spotsof fluorescent substances were observed under ultraviolet light. Although the chemical nature of the fluorescent substances is not yet
clear, certain inter-relationships were found between the distributionof the spots and the genome constitution of the available species.
Within the genus Tritz'cum, the pigment spots produced by one specie3
seem for the most part to correspond to those found in the others, andeven to increase in number with the increasing participation of different
genomes. Furthermore, spots produced by Agropyrum and Aegilops
are partially similar to those of Tritz'cum. Secale and Haynaldia
showed almost identical figures of pigment spots, in particular as regards the two major pigments. However, they differed markedly fromthose of Tritz'cum. Hordeum se~me:l to be quite different from allother examined genera.
46. Paper-chromatographic Analysis of Anthocyanins Occurring in

Several Varieties of Japanese Mornz'ng Glory (Pharbitis Nil)with Special Reference to the Role of the mg Allele(by Yuki hide ABE)

In No. 4 of the Annual Report, a general survey of coronal anthocyanins occurring in several varieties of Japanese morning glory. waspresented. It was po~nted out that the mg allele apparently plays the

RESEARCHES CARRIED OUT IN 195473

main part in the hydroxylation of the side benzene nucleus in theanthocyanin molecule.
In order to find further evidence in support of this view, several
experiments were carried out on eight strains having an mg or +mg
gene (Table). The anthocyanin pigments were extracted with 1%
methanolic hydrochloric acid, and the extracts were subjected to hydrolYSis with hot 10% hydrochloric acid. Then the crude solutions ofanthocyanidin were purified as usual and chromatographed with two
kinds of solvent mixtures: acetic acid/36% hydrochloric acid/water(5: 1: 5, v/v) and iso-propyl alcohol/ 5% hydrochloric acid (55: 45,v/v). The results are summarized in the Table:
Anthocyanidins occurring in various genotypes oj Pharbitis Nil.

Genotype
concerning
flower color

CorollaFlowertube

Epidermis2)

EpidermisI-+-m-g-+-Pr-+-c-a-+-c-+-,,-l)+!-B-5)+ mg pr + ca +c +"mg +P"+ca+c+,,
mg pr +ca+c+"+ mg pr + ca C +"mg pr +ca C +"+mg+P,,+ca+c r

P

IPI P C3J
9 44)

0

0
M 9
R 8
M:O
1

Stem

Axis ofseedling

Autumnal
leaf

Subepider- Subepider- Palisade
mal tissue mal tissue parenchym

I PI P C
PIPC: IPIPC:e,IPI0 5P C5 COl
9C082 CIO+-/II!

d 0 5 5 C 0 3 7 C064COO-Hll
1 ± C * + ± C 6 2 2 G631

6 4

2 ± C 8 2
0 0 !W 0 0
w1 0 00 0
W 10
0 -/II- +*
0 7

'wl

ci

± C 7 1 2!1!

d 7 2 1 ICl 0 0 -HlI
1

0 C02 8 C 0 6 41 CI 0 + -/II0 C 6 2 2 C 6 3 1 C++
1
3 G: 0 0 0 GOOO'GOOO*

mg pr +ca+c r I W 1* + 0* Yi-/ll- + 0* Gj 0 0 0 G j 0 0 0 I GI 0 0 0* containing anthocyanin pigment probably in the form of colorless
leucobase. 1) all homozygous. 2) showing localization of anthocyanins. 3) PI: pelargonidin, P: peonidin, C: cyanidin. 4) Relative
amount of anthocyanidins (in 10 parts). 5) B: bILle, G: green, M:
magenta, P: purple, R; red, W; white, Y: yellow, C: from red topurple.

As shown in the Table, pelargonidin does not occur in any tissueof the +mg plants, whereas it does occur in the mgplants. Moreover,
the action of the mg allele appears not only in the corolla, but also incertain vegetative organs, in several strains which are capable of producing anthocyanins. In order to test this hypothesis, further experi.

74

RE3EARCHES CARRIED OUT IN 1954

ments are in progress, using the progeny of the inter-hybrids between
the above strains.

47. Studies on Natural Anthocyanins(by K6z6 HAYASHI and Yukihide ABE)

Brilliant and attractive flower colors produced by anthocyanin pigments were sub~ects of interest of classical plant genetics. When thegenes controlling them were discovered, attempts were made to investigate the biochemical processes underlying pigment formation andcolor variation in relation to these genes. But, even today, a direct
approach to these fundamental problems is virtually impossible, since
we know too little about the complicated biosynthetic processes ofanthocyanin production in living plant cells.
In order to contribute something to our knowledge of the natureof plant pigments, we have studied anthocyanins occurring in various
plants. In 1954 the following investigations were carried out:a) Red coloring matter in the leaves of PerUla varieties.
The anthocyanin in the dark red leaves of Perilla frutescens var. crispa
was shown by K. KONDO (1932) to be a pentose-glyco~ide of delphinidin combined with protocatechuic acid, which he termed 'perillanin '.
C. KURODA and her co-worker (1935) arrived, however, at a different
conclusion, holding that the leaf pigment of that plant is not perillanin, but an esterified anthocyanin composed of cyanin (cyanidin
3.5-diglucoside) and p-hydroxycinnamic acid. They have given thiscompound the name of 'shisonin '. Our careful experiments carriedout by means of paperchromatography as well as by the usual methodof chemical analysis have shown that the pigment component in question is shisonin, as pointed out by KURODA. Our studies have also
disclosed that the red leaves of other garden varieties of the same
species owe their color to shisonin.
Reference: HAYASHI, K. and ABE, Y. 1955. Studies on Anthocyanins,
XXV. Paperchromatographic investigation on anthocyanins occurring in theleaves of Perilla varieties. Bot. Mag. (Tokyo), 68: 71.

b) Anthocyanin of purple-red flowers of Lespedeza.
Lespedeza, one of the beauties among the autumn plants in Japan,
includes several species. The deep purple-red flowers of L. Thunbergii
seemed to be the best material for the preparation of the pigmentcomponent. Mter several unfruitful experiments, w~ were able to75

RESEARCHES CARRIED OUT IN 1954

isolate a coloring matter in a pure crystalline state. This consisted ofdeep chocolatebrown needless having a brilliant greenish metallic lustre.
A series of chemical analysis have shown that the pigment is nothingelse than malvin, £.e. 3.5-diglucoside of malvidin. The same anthocyanin
occurs also in the flowers of several other species of Lespedeza; thiswas shown by paperchromatographic analysis. Finally, it is interesting
to note that the snow-white flowers of L. japon£ca contain an appreci.
able amount of the pigment in a colorless form, which is readily
converted into a malvidin derivative simply by treatment with warmhydrochloric acid. An extensive study concerning the mechanism oftransformation of this colorless substance into malvin in living cells
may throw some light upon the quite obscure biosynthetic process
leading to the formation of anthocyanin pigments in general.
Reference: HAYASHI, K. NOGUCHI, T. U. ABE, Y. 1955. Studien tiber
anthocyane, XXVI. Uber den Farbstoff der Blilten von Lespedeza Thunbergii.
Bot. Mag. (Tokyo), 68: 129.

c) Survey of anthocyanins responsible for the autumnal reddening of leaves in Japan. Fairly extensive investigations have been carried out on the reddening of autumn leavesof deciduoU3 trees chiefly in the neighbourhood of Nikko. Leavesof about 70 kinds of plants were examined by the paper-chromatographic
method. As has been previously suggested by foreign authors, theleaf anthocyanin consists, for the most part, of cyanidin monoglucoside,which is protably identical with chrysanthemin. This shows that inleaf tissues a pattern of biosynthetic reaction is preponderant, andthat this reaction brings about the formation of a Cs-Cs-Cs com·pound, in which the two terminal Cs-groups are substituted by aphloroglucinol- and a pyrocatechol-moiety, respectively. So far as theleaf anthocyanins are concerned, the intracellular processes giving riseto the formation of the above two moieties should provide an important clu'3 for our biochemical approach to the basic problem of pigment
production. A full account of our data will be published shortly.
L. CYTOLOGY AND GENETICS OF SOME LOWER ORGANISMS

48.

The Delayed Appearance of the Q-AnUgen TransformaUon £n
Salmonella(by Tetsuo lINO)

The change of the O-antigen of S. anatum from 10 to 15 is accom·

76

RESEARCHES CARRIED OUT IN 1954

panied by its lysogenization for bacteriophage €, and the phage itself
is regarded as the tranSforming principle of this change CISEK I and
SAKAI, 1953). This phage € also acts as a carrier of genetic elements
on transduction, according to ZINDER and LEDERBERG'S study (1952)
on another strain of Salmonella. Accordingly, when the cells of S.
anatum. are transduced by the FA (filtratable agent) of S. newington
which is lysogenic for €, they change their a-antigen from 10 to 15.
In general these changes can be detected simultaneously on the colonies which are developed on the plate, spread with the treated cellsof S. anatum. In the course of experiments designed to test thesimultaneous induction of transduction and a-antigen transformation,
it was found that the antigen transformation appeared later than thetransduction, as is to be described below.
A maltose- broth culture (l ml, 5 x 108 cells) of the streptomycin
sensitive CSS) strain A&-l of S. anatum was mixed with an equalvolume of cultural filtrate of the streptomycin resistant (Sr) strain 84
of S. newington. As a control, the same culture was mixed with thefiltrate which had been heated at lOoDe. They are incubated for 24hours more, and Sr cells were screened by spreading the culture on
media containing 5 mg/ml of streptomycin.
In three experiments, 122, 217 and 265 sr colonies respectively wereobtained from about 5 x 108 cells, but none from the control series.
The lysogenicity and a-antigen type were examined on these Sr colonies using the turbidity test and the slide agglutination test. All of
the Sr colonies obtained from the first two experiments showed lysogenicity and a-antigen 15, but some colonies obtained from the third
experiment showed only the change to lysogenicity, the a-antigen 10
remaining unchanged. Thirty of these colonies were picked up andspread on broth agar successively at intervals of 48 hours, and theantigen type of their offspring was tested. It was found that 20 to
60% of the random colonies contained cells showing antigen type 15
in each spreading, and the cells, which had acquired the 15-type antigen, maintained this property through further generations. In thecourse of this change the colony should be agglutin'lted by brothanti < > 10 serum and anti< > 15 serum. As a matter of fact, nocolony showed agglutination by both antisera. This fact is possibly
due to phenotypic delay of antigen transformation, and is interesting,since it may provide a convenient material for analyzing the processof the expression of the antigen transformation.

77

RESEARCHES CARRIED OUT IN 1954

49. The Paths of the Reversion in the Methionine-requIring Strainof Ustilago maydis(by Tetsuo lINO)

The reversion of the methionine·requiring strain of U. maydis 4-25
(Mi) takes place through at least two distinct paths; Mi--.CR and Mi--.IR--.IR' (Annual Report, No.4, 1953). The estimation of the frequencies of each step is important in order to certify the distinction
between the two processes. However, it is difficult to do this exactly,
because of the simultaneous occurrence of these changes in the same
culture, and of the similarity of the CR and IR' cultures. An approximate estimation, however, is possible if the frequency of IR--.IR' is
not too large in comparis~>n with the frequencies of other steps.
The rate of the change from CR to IR in the total reversion isvariable according to exr:eriments. It has been estimated that 25%
on the average (ranging from 0 to 38%) of the reversional mutant,when tested immediately after screening from a single colony by theminimal agar plate method, is CR. As reported previously (Annual
Report, No.3, 1952), the mutation rate of Mi to methionine independant is 3 x 10- 7, so the mutation rates to CR and IR are estimated tobe approximately 8 x 10- 8 and 2 x 10- 7 respectively.
For the estimation of the rate of the change IR--.IR', the usual
plate-screening method is inadequate, because the dense population on
the plate suppresses the growth of IR', and makes the comparison of
growth rates between the two types impossible. Accordingly, themaximum frequency has been estimated from the number of cultures
which contain IR' among a series of successive minimal liquid cultures of IR, considering the selective advantage of JR' over IR. The
result indicates that the frequency of the mutation from IR to IR' islower than 5xlO- 5• This value is low enough to elim:nate the pcssibility of the appearence of IR' in the single colony culture of Mi. In
other words, all the reversional mutants obtained by the minimal
plate screening method are regarded as one step mutants CR.
In parallel with these experiments, .the possibility of the appearanceof a conditional mutation in the course of reversion was examined by
testing the nutritional requirments other than methionine of reverted
strains which were obtained by spreading washed cells suspensions ofstrain 4-24 on vitamin.H 20 2 treated peptone (methionine-free) media.
All of the 75 mutants tested were found to be unaccompanied by any
nutritional requirements other than methionine. This result indicates'(8RESEARCHES CARRIED OUT IN 1954

that conditional mutation does not play important part in the reversion of the methionine requirement.

50. A Cytoplasmic Polyhedral Virus Occurring in the Silkworm(by Mitsuo TSUJITA)
It is well known that the virus of the polyhedral disease of thesilkworm invades the nucleus of epidermal cells, adipose tissue cells,
tracheal epidermal cells or haemocytes (blood cells), and produces
polyhedral crystals, ISHIMORI (1934) first found polyhedra occurringin the cytoplasm of the mid-gut epithelial cells. His observation has
been confirmed by LoTMAR (1941), TSUJITA (1949), XEROS (1952), and
SMITH and XEROS (1953). In order to know whether or not the virus
occurring in the cytoplasm is different from that multiplying in the
nucleus of the epidermal and some other tissue cells, I had been looking for larvae with mid-gut polyhedrosis. Recently I found materialof this kind, and carried out microscopical and electron microscopicalobservation and experiments with it. The results obtained may besummarized as follows:i) The mid-gut polyhedrosis of the silkworm shows symptoms
similar to that of flacherie disease, especially as regards the change
appearing in the alimentary canal. The mid-gut, especially its middle
and posterior portions, takes on a distinctly wh itish colour.
ii) Numerous polyhedral bodies are produced in the cylindricalcells, but never in the goblet or interstitial cells of the epithelium.
The polyhedra are produced only in the cytoplasm; the nucleus isentirely free from polyhedra or virus-like particles.
iii) The polyhedra are almost identical in shape with those produced in the nucleus.
iv) The sections of the polyhedra exhibit numerous holes or protuberances which are of about the same size as the spherical particles
which appear on treatment with dilute alkali. The polyhedra develop
from clusters of spherical particles included in a proteinous matrix.
These particles probably represent viruses.
v) These are two types of the polyhedral virus infecting the silkworm, a rod-shaped one and a spherical one. The former usually
invades the nucleus of the epidermis and some other tissues, while
the latter seems to invade specifically the cytoplasm of the cylindricalcells of the mid-gut epithelium.
Reference: TSUJITA. M. 1955. Cytoplasmic polyhedral virus infecting thesilkworm. Proc. Japan Acad. 31 (2): 93-98.

RESEARCHES CARRI:ED OUT IN "195479

51. Studies on the Mitochondrial Granules isolated from Paramecium caudatum(by Ky6z6 WATANABE, Mitsuo TSUJITA and Bungo SAKAGUCHI)

The cytoplasmic constituents of Paramecium caudatum wereseparated into four fractions, T, Mw, Pw, and 52, by the differential
centrifuge method.
In our experiment with caudatum, it was difficult to isolate completly
the mitochondrial granules from other large granules or smaller parti.
cles. However, according to our microscopical observations of each of
the fractions, it is certain that the mitochondrial granules are mainly
concentrated in the Mw fraction and the large part of the smaller
particles are precipitated in the Pw fraction.
Reference: WATANABE, K., TSUJITA, M. and SAKAGUCHI, B. 1955.
Studies on the mitochondrial granules isolated from Paramecium caudatum.
Bull. Educ. Fac. Shizuoka Univ. 5: 153-160.

52. Fine Structure of Mitochondria in Paramecium(by Mitsuo TSUJITA, Seizo TSUDA and Ky6z6 WATANABE)

The present paper deals with the results of an electron-microscopical study on the fine structure of mitochondria in Paramecium
carried out by means of ultra-thin sections.
The mitochondria lying under the peIficle and those scattered in
the cytoplasm show the same structure. Each mitochondrium iscovered with a membrane. In addition, there is a membranous fold
or a plicate structure in the interior of each mitochondrium. This
plicate structure does not always have the same appearance; it may
be indistinct or it may exhibit a network. These varieties in internal
structure seem to represent the different phases of their physiological
function.
The mitochondria occurring in the ectoplasm seem to have somerelation to the formation of trichocysts; also they may be somehow
related to the energy source for ciliary movement.
Reference: TsUJITA, M., WATANABE, K. and TSUDA, S. 1955. Finestructure of mitochondria in Paramecium. Electron microscope 4: (in press).

80

RESEARCHES CARRIED OUT IN 1954

53. Studies on the Lysogenicity of Pseudomonas solanacearum(by M. TsUJITA, C. MATSUI, S. TSUDA and O. YOSHIZAWA)

Two of the seven lysogenic strains of Pseudomonas solanacearum
E. F. SMITH isolated by N. OKABE, &-9 and T-c200, together withseveral indicator strains of the bacteria were used as materials, andthe results obtained are summarized below:a) Lysogenic strain T-c200. The lysogenicity of the strain
T -c200 is a stable hereditary character, and its cell progeny consists
wholly of lysogenic cells. The virus liberated from the strain is active
against P. solanacearum, Strain &-9, S-IX, E-3, A-8 and S, and someof these indicator strains can be lysogenized.
During the logarithmic growth phase of strain T-c200 in potatodextrose solution at 34°C, the increase of free virus is paralleled by
the growth of bacteria, and the ratio of bacteria-free virus isnearly constant, being about 5xl04/1 mI. It is assumed, therefore,that the bacteria which spontaneously produce mature viruses are asmall fraction of the whole bacterial clone.
Strain T -c200 and its free virus are killed by irradiation with ultraviolet light, and the ratios of inactivation of bacteria and virus arefound to be roughly the logarithmic function of the irradiation dose.
The proportion of strain T-c200 which produces mature virus isr.ot increased, but decreased, by irradiation with ultraviolet light;
therefore, strain T -c200 seems to be a non-inducible lysogenic strain.
After ultraviolet irradiation, bacteria without lysogenicity can beobtained from the survivors of the former colonies. The bacteria,
having lost their lysogenicity, are sensitive to their original virus,
but can be relysogenized. Thus, lysogenic bacteria may be cured
from their lysogenicity by ultraviolet irradiation. The loss oflysogenicity is never spontaneously reversed. Thus, this bacterial
character must have undergone a genetical change.
As for the mechanism of the loss of lysogenicity by irradiation
with ultraviolet light it is most probable that the prophage, i. e. theanlage of the phage, is inactivated.
Reference: TSUJITA, M., MATSUI, C., TSVDA, S. and YOSHIZAWA, O.
1954. Studies on the lysogenicity of Pseudomonas solanacearum. I. Lysogenic
strain T -c200. Virus 4 (4): 308-312.

b) Response types shown by the T-13 bacteria after

RESEARCHES CARRIED OUT IN 1954

81

exposure to 5-9 phage. Cells of a 1'-13 culture which were
exposed to 5-9 temperate phages showed the following five responses.
(0 Reversibility. The cell is unchanged, and given rise to acolony of 1'-13 cells that may themselves give any of the 5 responses.
Oi) Lysis (productivity). Virus multiplication occurs within thecell, resulting in lysis and the production of new free phages. (iii)
Lysogenicity. The infected cell gives rise to a colony containing
lysogenic cells. These cells spontaneously produce 5-9 free phages,
but cannot induce multiplication and maturation of phages by theirradiation of ultraviolet light. The cell is immune to 5-9 free phage.
(iv) Lethality. The cell is killed, and the absorbed phage is inactivated. (v) Resistance. The cell gives rise to a colony of nonlysogenic 5-9 resistant cells.
The proportions of the cells showing various responses change
according to the environmental conditions of the culture, such astemperature, or age of culture. When a culture kept for 18 hours
at high temperature (34°C) is infected with 5-9 phages, most of themshow a lethal response, and the remainder exhibit productive, reversible, and lysogenic responses. On the contrary, when the cells arecultured for 73 hours at the same temperature, 90% of the infected
cells show reversible resj:onse, and only part of them can produce
mature 5-9 phages.
Lysogenic cells can readily be produced at low temperature (25°C).
When cells cultured for 18 hours at the low temperature are infectedwith the phages, about 60 % of them show a lysogenic response, andthe others reversible response, while no cell producing free phages isfound. However, when the cells are cultured for 73 hours, almost all
the infected cells show reversible response and only a small portion
are lysogenized.
As mentioned above, by raising the temperature during culture,
the acquirement of a lysogenic property by the cells in the clone canbe suppressed completely or partially. It must be noted, however,that the temperature treatment has no effect on cells in an establishedlysogenic culture.
The cause of these differences in response of different cells, except
the 5th response mentioned above, does not seem to lie in any genetic
factor, but may be found in the initial physiological differences among
the cells, or in differences created by infection.

82

RESEARCHES CARRIED OUT IN 1954c) A. double lysogenic strain. By superinfection of S-9
lysogenic cells with T--e200 temperate phage which is closely related
to the carried prophage, the following three lysogenic strains wereobtained: (i) A strain producing 8-9 phage only; this strain seems
to have originated by reversible response. (ii) A new strain producing
T-c200 phage only; this strain has probably been originated by thesubstitution of the T-c200 prophage for the superinfected S-9 prophage.
(iii) A double lysogenic strain carrying both prophages, T-c200 and
8-9; this strain has the capacity to produce two types of phages, 8-9
and T-c200, and also a new-type phage. The production of this newtype phage from the doubly lysogenic strain should probably be
attributed to the recombination of the host specificity characters of
the parent phages, T-c200 and 8-9, which are serologically related.
Referance: TSUJITA, M. and MATSUI, C. 1955. A double lysogenic
strain of Pseudomonas solanacearum. Proc. jap. Acad. 31 (3): 180-185.

54. Studies on the Multiplication of Bacterial Virus affecting
Streptomyces griseus(by Seizo TSUDA)
There are several works by previous authors on bacterial viruses
attacking Streptomyces. The bacteriophage infecting Streptomyces
griseus was first described by BONNETT (1946). His statements wereconfirmed later by several other workers (SAUDEK and CoLINGSWORTH
1947, WOODRUFF et al. 1947, LANGLYKKE et al. 1950, '51, TERADA et al.
1951, AlSO and MABUCHI 1950, '51, '52, and others). These authors
gave some information concerning the size and shape of some Streptomyces phages, but little is known of the behavior of the virus after
infection.
This report deals with electron-microscopical studies on themultiplication of a bacterial virus, S-l actinophage, infecting Streptomyces griseus. As seen in Figs. 1 and 2, the virus is uniformly
tadpole-shaped.
The S. gri'seus culture was prepared under submerged conditionsfor 24 hours at 25°C. The phage had propagated until the plaquecounts cxceeded 107 per mI. The activity of the phage was then examined by means of quantitative plaque counts.
A thin watery growth was frequently observed in submerged cultureof the host cells infected by S-l phagE! sttain, while non-infected

83

RESEARCHES CARRIED OUT IN 1954

host cells showed normal growth.
Multiplication of actinophage insubmerged culture of S. grz'seus ateach stage was estimated by theplaque count method, though thelysis was not complete in thisstage.
In the present experiments, thelytic change shown in Figs. 1
and 2 took place. Mature phages
were liberated from the host cell,
together with its cytoplasmic inclusions, after the period of mulThetiplication of the phage.
host cell then underwent gradual,
lytic change which was apparent
first on the margin of the cell.
When the multiplication of actinophage ceased, the nucleus
showed a filamentous structure(Fig. 3). Many rod-shaped particles, approximately 30 m,u inwidth and 100 m,u in length, canbe seen in Figs. 1, 2, and 3. It
seems that these particles areconcerned with actinophage development.
It has been assumed that thebacterial virus includes many gentical units, and that the multiplication of the virus within the hostcell proceeds with some regularity.

55. Electron-microscopical Studies of Ultra-thin Sections in
Penicillium chrysogenum(by Seizo TSUDA)

The cytology of microorganisms involves a number of structuresand events which are beyond the resolution limit of the liget micro-

84

RESEARCHE, CARRIED OUT IN 1954

scope. In order to make them accessible to investigation, the materials
are cut into thin slices by the woe of sectioning techniques recently
developed for electron microscopy. Studies of th's kin:! have been
carried out by such workers as HILLIER et al., ROBINOIV, CHAPMAN
et al., SJOSTRAND et al., TSUJITA et al. and HIGASHI.
The present paper deals with some observations of the structureof vegetative mycelium of Penicillium chiysogenum, the producer ofpenicillin, which appe~rs in ultra-thin sections.
The strain of Penicillium chrysogenum had been cultivated in thesubmerged condition at 28°C. for 3 days. As fixative, 0.5% osmium
tetraoxide in phosphate buffer solution was used. A fixed mycelium
was dehydrated by passing through the alcohol series, and imbedded
in a mixture of 95 parts of n-butyl metacrylate and 5 parts of methyl
metacrylate with 2% benzoyl peroxide as catalyst. The material inthis plastic substance was cut into thin sections of about 0.1 f1' by an
ultra-microtome.
The cell wall of the mycelia is thick, and it has a tendency to
become somewhat separated from the cytoplasm. The mycelium
contains several nuclei which have a thin membrane. The resting
nuclei in the electron micrograph appear round or ellipsoidal. The
nuclei as well as cytoplasm show a loose filamentous inner structure.
A great amount of cytoplasmic mitochondria-like granules arescattered in the reticulated cytoplasm. The cytoplasmic granules aremostly spherical or oval. They are surrounded by a limiting
membrane, and the inner surface of the inner layer of the limiting
membrane exhibits parallel lamellae which seem to protrude into theinterior of the granules. Detailed studies on cytoplasmic mitochondrialike granules in bacteria and fungi carried out by MUDD et al.,HARTMAN and Lm and many others revealed cytoplasmic granules
as dense bodies possessing limiting membranes.
The observation of the process of nuclear dividsion is under way.
M. THEORETICAL STUDIES ON BREEDING

56. Some Considerations on the Problem of Secondary Selectz'on
in Self-fertilized Crop Plants(by Kan-Ichi SAKAI)
Certain Indian plant breeders have recently expressed the viewthat in self-fertilized crop plants such as rice or wheat, "secondary
selection" is effective for improving the yielding capacity, because

RESEARCHES CARRIED OUT IN 195485

there should be some genotypic segregation within the varieties ofsuch plants. Theoretical considerations of this problem follow:(a) To what extent can a variety established by hybridization behomozygous? Assuming that the yielding capacity is controlled bymany "polygenes", and mutation occurs with a certain frequency,
the degree of homozygcsity in lines selected from a hybrid population
will be formulized as follows :-Let the rates of mutation from A to
A' and from A' to A both be m, the frequencies of AA, AA' and A'A'
plants in the goth segregating generation (D g, H g and R g) will be,1

Dg=2m(1-m)2Dg-l+THg-l+m2Rg-l,1
H g=2m(1-m)Dg- 1 +~2-Hg-l +2m(1-m)Rg- 1 ,and

Rg=m2Dg_l+-l-Hg-l+Cl-m)2Rg-l'

From these formulas, the frequency of plants homozygous for N pairsof genes will be found to be(D+R)g=;i- r }N.

f 1-(

t

2m

1+4m

Numerical calculations with these formulas show that, for instance,
when m=IQ-S and N=50, a line of FlO will be heterozygous for a genewith a probability higher than 18%.
(b) Mutation in pure lines: The following formula shows how avariety may become heterozygous as the result of mutation when it
was a pure line in a given generation, assuming that mutation occurs
in one direction only, with the freqlency of m'.
The frequency of desirable genes in the goth generation will thenbe
N g=No(l-m ' )g.
It will be found by using this formula that the loss of desirable
genes occurs at an extremely low rate; for instance, when m ' =10- s,
the proportion of genes to be lost after 20 to 50 generations is only
0.02 to 0.05.
It may be said in conclusion that "secondary selection" can beeffective in varieties bred by hybridization methods, but not so effective in varieties established from native varieties by the pure line
selection method. In this connection, the writer has proposed an improved system of stock seed propagation.

86

RESEARCHES CARRIED OUT IN 1954

57. Theoretical Basis for DiscriminaUng the Most Desirable
Hybrza Combinations in Early Generations(by Kan-Ichi SAKAI)

It has been suggested by several plant breeders that the potentialityof a hybrid population to segregate superior yielders in later generations can be predicted by an examination of the yield in the F z or F 3.
But some others are skeptical about this idea. In this paper a theoretical approach will be made to this problem.
First, let us consider the simplest case in which a character to be
selected is determined entirely by genes with additive effects andthere is no environmental variation. In this case, the mean value ofa hybrid population will be the additive genetic mean and the meansquare will provide an estimate of the additive genetic variance. If
individuals of such a population are distributed normally regarding
the measurement x, with the mean at X and with variance u2, we
can transform x to a variate u, with unit variance and the mean atzero, as u=(x-x)!u z. When a definite proportion of superior individuals, A, are selected, the selection differential 1 can be written as

1= ~ roo u~_ e-!u'du=~
A JU-ul -V27T
A.
where z is the ordinate of the unit normal curve at the deviate uf •Since z is determined by the value of A, the value of I can be foundfrom the value of A. The value of I on the x scale can be found by
multiplying 1 by u. The average value of superior individuals in thei-th population may then be given by Yi=xi+uil.
Next, we turn our attention to a more complicated case in which
intra-allelic interaction of genes causing heterosis and environmental
variation interferes with the additive gene effect. In this case wehave to subtract both the non-additive effect of genes and the effectof environmental variability from the observed values. The meanvalues of parents and hybrid populations may be written as follows:
1 -2(PI +Pz)=MP
F1=MP+h1
F 2=MP+ h

T1
F 3 =MP+hT(M? is the mid-parent value; h is the value of F 1 from MP.)RESEARCHES CARRIED OUT IN 1954

87

The value of MP found from these formulas may be regarded as an
estimate of the additively genetic mean of the given hybrid population.
The additive genetic variance may be estimated by the method ot'
partitioning variance components of Mather (1949). For practical
purposes, observation of three hybrid populations, Fl. F 2 and F 3, andthe parental lines will be recommended. By using those formulas,
we can estimate the values of D, Hand E, which are variance components attributable to additive gene effect, non-additive gene effectand environmental variation, respectively. From the value of D, we
can estimate the value of Y for a given hybrid combination (i) as
follows:
Y,·=MP+lYDi=MPi+ 1YVFa-2VF.-Vp(F,) .

In a further complicated case in which intergenotypic competition
influences the character, additional breeding of F 3 lines and F 4 bulk,
and if possible, both back-cross populations, will be needed. Formulasfor various hybrid populations, taking competitional variance into account are as follows:

VF.= ~ D+ ~ H+C"+-tG"+E
3

3

3

3

VFa= T D +-i6H +-2- C"+-S-G"+E
VFa=+D+{6 H+ ~

- 7 D +64
7H +T
7 C" +32
7 G"+E
VF'-g

VB,+VB'=2~D+ ~ H+C"+G"+2E
Vp,VF,=E(e" and G" stand for variance components due to additive
and non-additive effects of competition, and Nis the numberof plants within line.)
The value of D will be found from these formulas and Y may be
estimated as before.
Comparison of Y values among a number of hybrid populations
will show which of them are more promising for the breeder thanothers.

BOOKS AND PAPERS PUBLISHED IN 1954
BY STUFF MEMBERS
ENDU, T. Biochemical and genetical investigations of flower color in Violatricolor L. I. Interrelationship of pigment constituents occurring in ten
varieties. lap. lour. Bot. 14 (2): 187-193.
FUJII, T. Studies in chlorina mutants of Einkorn wheat induced by X·rays.
(Abstr.). lap. lour. Genet. 29 (4): 153 (in lap.).
GOTOH, K. Genetic studies on egg plant (Solanum melongena L.). III. Further
investigations regarding the degree of heritabiIiiy and the number ofeffective factors. lap. lour. Genet. 29 (3): 89-97.
Inheritance of doubleness in Zinnia elegans L. lap. lour. Breed. 4 (1):37-40 (in lap.).
The concept of .. selection limits" and breeding. Agric. and Hort. 29 (8) :981-985 (in lap.).
HAYASHI, K. Methods of Experimentation on Plant Pigments. (Handbook of
Biological Experiments 6, C) pp. 72 (in lap.). Nakayama Shoten, Tokyo.
- - , T. NOGUCHI and Y. ABE. Studien tiber Anthocyane, XXIV. Keracyanin,
ein Farbstoffprinzip in den feuerroten Bltiten von Canna generalis. PILar·mac. Bull. 2(1): 41-45.
Uberblick der Arbeiten tiber Anthocyane in lapan, unter besonderer Be·
rticksichtigung der nattirlichen Pflanzenfarben. Pharmazie 9 (7): 584-588.
KOMAI, T. Seventy·five years of lapanese zoology: Genetics. Zoo I. Mag. 63(8/9): 355-357.
An actual instance of microevolution observed in an insect population.
Proc. lap. Acad. 30 (10): 970-975.
Heredity and Family Records. 2nd. ed. pp. 261 (in lap.). Hokuryukan,
Tokyo.
MAKINO, S. and T. H. YosIDA. Breeding experiments of white rats and mice,
III. Notes on the abnormal characters found in some inbred strains ofwhite rats and mice. Misc. Rep. Yamashina Inst. Ornithol. & Zoo I. 4:
141-145 (in lap.).
MATSUMURA, S. Chromosome analysis of the Dinkel genome in the offspringof a pentaploid wheat hybrid. V. Gigas-plants in the offspring of null·isomic dwarfs and analysis of their additional chromosome pair. Cytol·
ogia 19(4): 273-285.
Gene Analysis. 1. Morphological characters. in KIHARA's .. Studies on
Wheat ", Rev. ed. 264-390; 2. Physiological characters (with K. YAMA·SHITA). Ibid. 391-434; Induced mutation (with K. YAMASHITA). Ibid.
615-681. (in lap.). Y6kendo, Tokyo.
Cytogenetics and Breeding of Wheat. 2nd. ed. pp. 247 (in lap.). Y6kendo,
Tokyo.

BOOKS AND PAPERS WRITTEN BY STUFF MEMBERS•89

Mechanism of translocation, in KIHARA's .. Cytogenetics". 1. Rev. ed.,107-123 (in lap.). Yokendo, Tokyo.
Nullisomic dwarn! and their gigas-plants in the offspring of a pentaploid
wheat hybrid. Rapports et communications, VIlle. Congres Intern. de
Botanique, Paris 1954, Sect. 9 & 10: 196-197.
- - and T. FuJII. Irradiation experiment with ultra-short wave in Einkorn
wheat. lap. lour. Genet. 29 (1): 13-17 (in lap.).
NAWA, S. and T. TAIRA. Pterins found in silkworm and Drosophila. Proc. lap.
Acad. 30 (7): 632-635.
- - and - - . The relationship between eye pigment and pterins of D. me/anogaster. D. I. S. 28.
- - et a/. Studies on pteridines. VI. The pterin from Bombyx mori (silkworm)
and Rana nigromacu/ata (frog). lour. Biochem. 14: 657.
OGUMA, K. Studies on the effect of heredity vs. environment on determinationof characters of organisms. Science Bulletin 6 (10): 316-322 (in lap.).
OKA, H, Classification of rice varieties by intervarietal hybrid sterility (Phylogenetic differentiation of the cultivated rice plant. II. lap. lour. Breed.
3 (3/4): 1-6 (in lap.).
Varietal variation of the responses to day·length and temperature and thenumber of days of growth period (Phylogenetic differentiation of thecultivated rice plant. III.). lap. lour. Breed. 4 (2): 92-100 (in lap.).
Intervarietal variations in the response to fertilizer in rice. (Phylogenetic
differentiation of the cultivated rice plant. IV.). lap. lour. Breed. 4 (2):101-110 (in lap.).
Phylogenetic differentiation of the cultivated rice plant. V. Variation ofminimum germination temperature and temperature constant among rice
varieties. lap. lour. Breed. 4 (3): 140-144. (in lap.).
Studies on tetraploid rice. II. Individual variation in tetraploid rice. Jap.
Jour. Genet. 29 (1): 18-25 (in Jap.).
Studies on tetraploid rice. III. Variation of various characters among
tetraploid rice varieties. Jap. Jour. Genet. 29 (2): 53-67 (in Jap.).
Studies on tetraploid rice. IV. Fl hybrids between tetraploid varieties ofrice. Jap. lour. Genet. 29 (3): 101-108 (in Jap.).
Intervarietal variation in the responses to temperature and day-length inrice. Agric. and Hort. 29 (4): 485-490 (in Jap.).
SAKAI, K. New topics in rice breeding. Agric. and Hort. 29 (1): 95-98 (in lap.).
A theoretical note on the problem of secondary selection in autogamous
crop plants. Agric. and Hort. 29 (2): 241-244 (in lap.).
A plan for the production of superior seed in cereal crops. Agric. andHort. 29 (9): 1103-1105 (in lap.).
Theoretical studies on the technique in plant breeding. I. Value of heritability in bulks and plant progenies of successive hybrid generations in
autogamous plants. Jap. Jour. Breed. 4 (3): 145-148 (in Jap.).

90

BOOKS AND PAPERS WRITTEN BY STUFF MEMBERS- - and K GOTOH. The competitive ability of Fl hybrid of barley. lap. lour.
Breed. 3 (3/4): 98 (in lap.).
TAIRA, T. and C. 6sIMA. Population genetics on the dimorphism in color
patterns in Drosophila rota. D. I. S. 28.
TAKENAKA, Y. Cytogenetic studies on Nicotiana. IV. La Kromosomo 17-19:
706-713 (in lap.).
- - Cytogenetic studies on Nicotiana. V. Bot. Mag. 66: 269-276 (in lap.).
- - and T. TATEOKA. On the origin of Prunus Yedoensis. La Kromosomo
21: 777-778 (in lap.).
TANAKA, Y. Unstable genes, a comprehensive review. Proc. Annual Meet.
Tokai Branch Seric. Soc. lap. 2: 29-35 (in lap.).
TATEOKA, T. On the systematic significance of starch grains of seeds in Poa·ceae. lour. lap. Bot. 29 (11): 341-347 (in lap.).
- - Karyotaxonomy in Poaceae. II. Somatic chromosomes of some species.
Cytologia 19 (4): 317-328.
TSUJITA, M., S. TSUDA and K WATANABE. Electron-microscopic study oninternal structures of Paramecium. Kagaku 24 (3): 150-151 (in lap.).
- - , - - and - - . Studies on the internal structures of Paramecium caudatumby means of ultra-thin sections. Rep. Sci. Stud. of Dept. Educ. Sizuoka
Univ. 3: 81-90 (in lap.).
- - , C. MATSUI, S. TSUDA and O. YOSHIZAWA. Studies on lysogenicity of
Pseudomonas solanacearum E. F. Smith. I. Lysogenic strain T·c 200. Virus
4 (4): 308-312 (in lap.).
TSUDA, S., M. TSUJITA and C. MA'ISUI. Electron-microscopic studies on internal structure of Aspergillus oryzae. Kagaku 24 (8): 417-418 (in lap.).
YOSIDA, T. H. Tetraploid chromosome constitution characteristic of the tumor
cells of the TAKEDA sarcoma. Gann 45 (1): 9-15.
Karyological studies of the T AKIZAWA qUinone-carcinoma and the EHRLICH ascites cercinoma in mice. Cytologia 19 (2/3): 225-238.
Karyological study on the MY mouse carcinoma and sarcoma. Gann 45(2/3): 162-164.
Individuality of the chromosomes in the tumor stern-cells of the TAKIzAW A
quinone-carcinoma of mice. Zool. Mag. 63 (1): 18-21 (in lap.).
Karyological characteristics of the tumor cells of the EHRLICH ascites
carcinoma. Zool. Mag. 63 (1): 22-25 (in lap.).
- - and S. MAKINO. Breeding experiments of white rats and mice. IV. Misc.
Rep. Yamashina Inst. Ornithol. & Zool. 5: 185-193 (in lap.).

i

.
i

m~fi!¥lVfl9Lm~3t~¥*

m5~

1!g;fl:l30:,¥ 9 Jj 28 B I'P!PU
1!g;fl:l30 ~ 9 Jj 30 B ~fi

~IT~
t-

/J\
lt~
~'J!.
IEft)Jt~~iJf:reiYi'F'::I~\iU~ ~

~-

i!*
1.

!Ii

1fiJ;j:j:

OO:ft~~~liJf~F.JfP3

I'IJ !PU ~

'i"

a:J

5t

JICijCm fIlS S ~ liIi 1 11) 1
I'P.lPU F.Jf ~Jt~t:!: "- :7 Jv rt:!:JlOjem r!!5 S ~ iifi 1 11) 1

96 IT m~:iLiI~:J:jJf~BT

iPl/QI.!IiJ::=,I;1I;1fHHE 1,111m • (=,1;11;) 771, 772

o

.I.

400

•,

600
.

800
."

1000m
,