NATIONAL INSTITUT~ OF G~N~TICS
JAPAN

ANNUAL RI:PORT
No. 11

1960

Published by

THE NATIONAL INSTITUTE OF GENETICS
Misima, Sizuoka-ken, Japan
1961

Annual Reportof theNational Institute of Genetics\

I

No. 11, 1960!

)•
Published by

The National Institute of Genetics, Japan
1961

CONTENTS
General statement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Abstracts of diary for 1960
Staff
Council............................................................
Projects of research for 1960
Joint researches supported by the grant from The Rockefeller Foundation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Foreign visitors in 1960 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Researches carried out in 1960

A.

Genetics, cytology and biochemistry of animals
1. The persistence of induced lethal genes in experimental populations (OSHIMA and KITAGAWA)
2. The persistence of deleterious genes in natural populations ofDrosophila melanogaster (OSHIMA and KITAGAWA)............
3. Heterozygous effects of natural lethal and semi-lethal chromosomes on pre-adult viability (OSHIMA)
4. The persistence of some natural lethal chromosomes in experimental populations (OSHIMA). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5. Selection experiment on migrating activity of Drosophilamelanogaster (NARISE and SAKAI)
6. The effect of inbreeding on migrating activity of Drosophilamelanogaster (NARISE and SAKAI)
7. Estimation of genetic parameters in dairy cattle (YAMADA). . . .
8. Variance increase of chaeta numbers in the heterozygotes ofDrosophila melanogaster (YAMADA)
9. Heterosis and reciprocal cross differences in egg production insome breed matings (KAWAHARA)
10. Influence of heterosis on hatchability of F 1 hybrids between
two breeds of the domestic fow 1 (KAWAHARA)
11. Negative correlation between rate of development and female
fertility in Drosophila melanogaster (HIRAIZUMI)
12. Some properties of the" sex-ratio" agent in Drosophila (SAKAGUCHI and DONALD F. POULSON)
13. A new case of spontaneous "post-axial polydactylism" in thehouse mouse (YOSIDA and SAKAMOTO)
14. Invasion of tetraploid cells from a common strain of YOSHIDA
sarcoma (YOSIDA).. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1
2
2
4
4

7
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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

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15.
16.
17.
18.
19.

20.

21.
22.
23.
24.

25.
26.
27.

28.
29.
30.
31.

B.

Invasiveness of hypertetraploid tumor cells from a hyperdiploidstrain of Ehrlich ascites carcinoma (YOSIDA). . . . . . . . . . . . . . . .
Chromosome condition of hypertriploid Hirosaki sarcoma (TANAKA and YOSIDA)..........................................
Alteration of cell populations in serial transplantations of hypotetraploid Ehrlich carcinoma (MATANO)
Proliferation of Ehrlich ascites carcinoma cells in vitro (UTSUMI
and YOSIDA)_. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Studies on mouse leukemia I.
Incidence of leukemia in
AKR/Jax strain mice bred in Misima (YOSIDA, KURITA andSAKAMOTO)
Studies on mouse leukemia II. Susceptibility to radiationinduced leukemias in various substrains of mice bred in
Misima (KURITA and YOSIDA)
Studies on mouse leukemia III. Chromosomes in spontaneousand induced mouse leukemias (KURITA and YOSIDA)
Susceptibility of rats to tumor induction by treatment withmethylcholanthren (YoSIDA and KURITA)
Maternal effect of +lem gene on pterine reductase (TSU.TITA)..
Genetical and biochemical studies on the metabolism of pteridines in silkworm-The structure of the yellow pigment(NAWA)....................
Biochemical studies on Drosophila pteridine reductase (TAIRA
and NAWA)
Phosphatase activities in the skin of hairless mice mutants(MORIWAKI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Comparison of the DNA-content of diploid and tetraploid
Ehrlich ascites carcinoma cells according to microspectrophotometer measurements (UTSUMI and YOSIDA)
DNA-content of Ehrlich ascites carcinoma cells with specialreference to its variation during mitosis (UTSUMI). . . . . . . . . .
Two factors influencing sensitivity to x-rays in the synthesisof actin during early embryonal stages (OGAWA)
Biochemical relation between the synthesis of actin and myosinin the regenerating limb tissue of Triturus (OGAWA)........
Effects of glucuronic acid and related compounds on the synthesis of skeletal muscle proteins in early Triturus embryo(OGAWA and KARUBE)

25
26
27
27

28

29
30
30
31

32
33
34

34
35
37
38

40

Genetics, cytology and biochemistry of plants
Genome analysis in the genus Oryza II. (KIHARA, NEZU, KATA-

32.

YAMA, MATSUMURA and MABUCHI)

40

CONTENTS
33.
:34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.

Floating habit of 10 strains of wild and cultivated rice (KIHARA,
KATAYAMA and TSUNEWAKI)
Production of polyploid wheat by nitrous oxide (KIHARA and
TSUNEWAKI)................................................
F 1 monosomic analysis of Triticum macha (TSUNEWAKI and
KIHARA)
Monosomic and conventional analyses of glume hairiness andear density in 8 varieties of common wheat (TSUNEWAKI) ..
Monosomic analysis of synthesized hexaploid wheats (TSUNEWAKI)
Marker lines in Triticum aestivum var. S-615 (TSUNEWAKI)....
An early ecotype of Agropyron tsukushiense var. transiens
Ohwi (SAKAMOTO)
Photoperiodic responses of Oryza species III (KATAyAMA)......
Studies on the intercellular spaces in rice (KATAyAMA)........
On the mechanism of the appearance of gigas-plants fromnullisomic dwarf wheat (OHTA and MATSUMURA)
Cytological and histological observations on male sterility insugar beets (OHTA and MATSUMURA) . . . . . . . . . . . . . . . . . . . . . . . .
Variation in susceptibility to blast fungus in cultivated andwild rice (KATSUYA)"
Variability of seed size in wild rice populations of Burma andsome African countries (SAKAI, lYAMA and NARISE).... . . . . . .
Comparative studies on growth rate of seedlings in wild andcultivated rice (SAKAI and lYAMA) . . . . . . . . . . . . . . . . . . . . . . . . . .
Seed growth in cultivated and wild rice (NARISE and SAKAI) . .
Survey of variation between Oryza breviligulata and O. glaberrima collected from West Africa (MORISHIMA and OKA) ....
Parallel variation from wild to cultivated forms in Sativa andGlaberrima series of Oryza (MORISHIMA and OKA)
Further studies on hybrid sterility relations among cultivatedand wild strains of rice (HINATA and OKA)
Hybrid swarms between wild and cultivated rice species, Oryza
perennis and O. sativa (OKA and CHANG)....................
A survey of drought resistance in wild and cultivated ricestrains (MORISHIMA and OKA)
Comparison of growth and fertilizer response between Oryza
sativa and O. glaberrima (HINATA, MORISHIMA and OKA) ....
Inheritance of mono and polyembryony in Citrus (FURUSATO). .
Breeding of amphidiploids from a hybrid between Nicotiana
rustica and N. tab4cum (FURUSATO) . . . . . . . . . . . . . . . . . . . . . . . .
Selection for low nicotine content (FURUSATO and MIYAZAWA)..

iii

41
42
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51
53
54
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55
56
57
57
58
59
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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

iv

Heterotic vitality in natural populations of Trillium kamtschaticum Pall (HIRAIZUMI, NARISE and FUKUDA)
58. Cytogenetic studies in the genus Nicotiana XIII (TAKENAKA) . .
59. Developmental studies in the genus Oryza II. Further studies
on embryo sac formation (DOIDA) . . . . . . . . . . . . . . . . . . . . . . . . . .
60. Cytological studies in the genus Polygonum II (DOIDA)........
61. Developmental studies in the genus Polygonum IV. Further
studies on the effect of gibberellin on microsporogenesis inthree Polygonum species (DOIDA)
62. Studies on flower color variation due to anthocyanins (ENDO)..
63. Anti-cancer activity of Gentiana extract (TAKENAKA, OGAWA,ODASHIRO and KARUBE)
57.

62'
63
64
65

66
67
69

Mathematical genetic

C.

64.
65.

66.

D.

An example of genetic system in which the gene frequency
equilibrium is stable under negative overdominance (KIMURA)
Mutational load when the phenotypic effect of mutational
damage is partially suppressed by developmental homeostasis(KIMURA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Population dynamics of selfing (MUKAI)

70

71
72

Genetics and biochemistry of microorganisms
67.

Transductions between curly flageller mutants in salmonella(IINO). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
68. A non-flagellated mutant which produces flagellar protein insalmonella (IINO and HARUNA)..............................
69. Host-range mutants of chi-phage (SASAKI) . . . . . . . . . . . . . . . . . . . .
70. The composition of ribonucleic acid in Yeast irradiated withultraviolet light (NAwA).. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
71. Nuclear structure of yeast (YONEDA). . . . . . . . . . . . . . . . . . . . . . . . . .
72. The effect of nucleoside supplementation on mutation (NAKAI)
73. Variations in the number of micronuclei found in the wild
populations of Paramecium polycaryum (MuRAMATsu)

E.

73
74
75
76
77
78
79

Radiation genetics in animals
74.

75.

76.

Two types of dose-rate dependence of radiation-induced mutation rate in spermatogonia and oogonia of the silkworm(TAZIMA, KONDO and SADO)Frequency of accumulated recessive embryonic lethaIs in a
popular commercial race, Cll5, of the silkworm and theirspontaneous mutation rate (TAZIMA and KOBAYASHI)
Sensitivity and time of degeneration of spermatocytes irradiated

81

82

77.
78.
79.

80.
81.

82.
83.
84.
85.
86.
87.
88.
89.

CONTENTSv

at different stages of meiosis of the silkworm (SADO)
Reduction in number of sperm after irradiation of male silkworms (SADO)
Differences between times of death of the F 1 after irradiationof oogonia or mature oocytes (TAZIMA and ONIMARU)........
Recessive lethal mutations induced in mice by chronic irradiation during the whole development in three successive
generations (SUGAHARA, TUTIKAWA, MURAMATSU and TAKEDA)..
Shift of secondary sex ratio among the progeny of chronically
irradiated male mice (F , generation) (TUTIKAWA)............
A radiation genetical consideration concerning the genetic
structur of natural populations in Drosophila melanogaster(MUKAI)
Radiation-induced polygene mutation rate (MUKAI)............
Frequency of chromatid breaks at various doses of X- and rrays (YOSIDA, TAKAHASHI, MATANO and UTSUMI)
Effect of DNP (2, 4-dinitrophenol) on the frequency of X-ray
induced chromatid breaks in Ehrlich tumor cells (MATANO). .
Effect of sodium azide (NaNs) on X-ray induced chromatid
breaks (UTSUMI)............................................
Changes of ATP content in Ehrlich tumor cells after treatment with DNP and NaNs (MORIWAKI)
Cytological observations of ascites tumors treated with chronic
r-radiation (YOSIDA and TAKAHASHI) . . . . . . . . . . . . . . . . . . . . . . . .
A theory for the frequency distribution of cluster mutants(KONDO)A biophysical consideration on radiation mutagenesis (KONDO)..

84
84
85

86
88

89
90
91
91
93
94
95
96
97

F. Radiation genetics in plants
90.
91.
92.
93.
94.
95.

Radiation effects of beta- and gamma-rays in Triticum mono97
caecum (MATSUMURA)
Segregation ratio and viability of several chlorophyll mutantsin einkorn wheat (FUJII)
98
Preliminary experiments on the relation between dose rate andradiation effect in einkorn wheat (FUJII)
99
Relation between radiation effects and dose rates in rice
(MABUCHI, FUJII and MATSUMURA)
100
Effects of radiation on the susceptibility of wheat and riceseedlings to leaf rust (KATSUYA). . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Calculation of absorbed dose delivered to wheat seeds soaked
in 32p and 131 1 aqueous solutions (Kmmo and ISHIWA)
101

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO, 1 J

96.
G.

The effect of X-irradiated pollen grains on the formation of
empty hybrid seed in Nicotiana (TANAKA)........ . . . . . . . . ..

104

Technicalnote
97.

A preliminary calculation of depth dose curve for 60Co r-ray
irradiation (KONDO and ISHIwA)'.,

Books and papers published in 1960 by staff members

,...

105
107

GENERAL STATEMENT
In this year of 1960, a new department for human genetics wasestablished. Dr. E. Matsunaga became the head of the department. He was formerly professor of legal medicine at Sapporo
Medical College, Hokkaido. With the establishment of the new
department, our Institute has now seven departments. The new
department will be devoted to the studies on blood groups inrelation to natural selection as well as the studies of cytogenetics
in man. Dr. Y. Hiraizumi came back from U.S.A. after 4 years
study in Wisconsin and joined the staff of the department.
On November 7-8, we held a symposium on the effects of radiation in Misima. 72 persons attended the meeting.
The fourth summer seminar on genetics for the senior highschool teachers was well attended this year.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

ABSTRACT OF DIARY FOR 1960
Feb. 19. 81st meeting of Misima Geneticists' Club.
March 18. 82nd meeting of Misima Geneticists' Club.
April 15. 83rd meeting of Misima Geneticists' Club.
May 21. 31st Biological Symposium.
84th meeting of Misima Geneticists' Club.
29. 32nd Biological Symposium.
June
9. Meeting of all Japan Poultry Breeding Associations.
15. Meeting of Tobacco Research Workers.
July 17. 85th meeting of Misima Geneticists' Club.
20-23. 4th Summer Seminar on Genetics.
Aug.
8. 33rd Biological Symposium.
12. 34th Biological Symposium.
Sep.
13. 35th Biological Symposium.
17. Memorial Lecture and Movies (in Sizuoka).
30. 86th meeting of Misima Geneticists' Club.
Oct.
5. 36th Biological Symposium.
7. 37th Biological Symposium.
21. 87th meeting of Misima Geneticists' Club.
Nov. 7-8. Symposium on genetic effects of radiation.
25. 88th meeting of Misima Geneticists' Club.
Dec. 12. 38th Biological Symposium.
28. 89th meeting of Misima Geneticists' Club.

STAFF
Director
Hitoshi KIHARA, D. Sc., M.J.A.

Members
1.

2.

Department of Morphological Genetics:
Yataro TAZIMA, D. Ag., Head of Department
Motoo KIMURA, Ph. D., D. Sc.
Toshihiko SADO and Kimiharu ONIMARU
Department of Cytogenetics:
YO TAKENAKA, D. Sc., Head of Department
Tosihide H. YOSIDA, D. Sc.,

STAFF

3

Kazuo MORIWAKI, D. Sc., Tsuguo TATEOKA, D. Sc. (in Canada) andYoshiaki YONEDA.
3. Department of Physiological Genetics:
Chozo OSHIMA, D. Sc. Head of Department
Hitoshi KIHARA, D. Sc., M.J.A., Toshifumi TAIRA, Koichiro TSUNEWAKI
and Sadao SAKAMOTO (in U.S.A.)
4. Department of Biochemical Genetics:
Mitsuo TSUZITA, D. Ag., Head of Department.
Yoshito OGAWA, M. D., Saburo NAWA, D. Sc., Toru END6, Bungo
SAKAGUSHI (in U.S.A.), Tetsuo IINo, Ph. D. and Seizo TSUDA, D. Ag.
(in U.S.A.)
5. Department of Applied Genetics:
Kan-Ichi SAKAI, D. Ag., Head of Department
Hikoichi OKA, D. Ag.,
Yukio YAMADA, D. Ag., Akira MIYAZAWA, Takatada KAWAHARA andShinya IYAMA, D. Ag.
6. Department of Induced Mutation:
Seizi MATSUMURA, D. Ag., Head of Department
Sohei KONDO, D. Sc., Kiyoshi TUTIKAWA, Taro FUJII and Terumi MUKAI
Ph. D.
7. Department of Human Genetics:
Hitoshi KIHARA, D. Sc. Head of Department
Yuichiro HIRAIZUME, D. Sc.

Part-time Staff and Research Associates
Yoshinari KUWADA, D. Sc., MJ.A., Emeritus Prof. of Kyoto University
Kan OGUMA, D. Ag., Ex-Director, Emeritus Professor of Hokkaido University
Yoshimaro TANAKA, D. Ag., D. Sc., MJ.A. Emeritus Prof. of Kyushu University
Taku KOMAI, D. Sc., M.J.A. Emeritus Prof. of Kyoto University
Saziro MAKINO, D. Sc., Professor of Hokkaido University
Kazuo FURusATo.

Research members under the Grant from the Rockefeller Foundation
Rice Research Group:
Hiroko MORISHIMA, Keizo MTSUYA, Yukio DOIDA, Tadao KATAYAMA,
Kokichi HINATA and Masayuki SHINOHARA.
Animal Genetics Group:
Hiromi ISHIWA, Yoshikazu MATANO and Kazuhiko UTSUMI.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

Department of Administration
Kunio SHIMIZU, Head of Department
Katsutaro ANno, Chief of General Affairs Section
Toichi YOSHIKAWA, Chief of Finance Section
Hiroko NAKANO, Naomi MATSUBARA and Keiichi KAWASHIMA
Clerks, Typists, Chauffer, Field Laborers and Janitors...... 8

Misima Branch of Hatano Tobacco Experiment Station...... 5

Masao TANAKA, Head Assistant

Association for Propagation of the Knowledge of Genetics
Hitoshi KIHARA, President
YO T AKENAKA, Managing Director
Yataro T AZIMA, Managing Director

COUNCIL
Yo K. OKADA, Director of National Science Museum, Chairman
Kan OGUMA, Ex-Director, Emeritus Professor of Hokkaido University
Toshinobu ASAI, Professor of Tokyo University
Tanemoto FURUHATA, Director of Scientific Research Institute of Police
Hideo MATSUKUMA, President of Japan Monopoly Corporation
Toshitaro MORINAGA, Director of National Institute of Agricultural Sciences
Toshio SAITO, Governor of Sizuoka Prefecture
Takeo SAKATA, President of T. Sakata Company
Bungo WADA, Professor of Tokyo University
Yoshichi ASAMI, Emeritus Professor of Tokyo University
Seiz6 KATSUNUMA, President of Nagoya University
Riichi KAWAKAMI, Head of Department of Biometry, National Institute of
Public Health
Daigoro MORIWAKI, Professor of Tokyo Metropolitan University
Yakichi NOGUCHI, Professor of Tokyo University
Kenpo TSUKAMOTO, Director of National Institute of Radiological Sciences

PROJECTS OF RESEARCH FOR 1960
Department of Morphological Genetics
Genetics of the silkworm (TAZIMA)
Studies on food preference of the silkworm (TAZIMA)PROJECT OF RESEARCH FOR 1960

5

Radiation mutagenesis in the silkworm (TAZIMA and ONIMARU)
Cytological study of silkworm germ cells (SADO)
Genetic effects of chronic r-irradiation upon the silkworm (TAZIMA and
ONIMARU)
Theoretical studies of population genetics (KIMURA)

Department of Cytogenetics
Cytology and genetics of tumors (YOSIDA)
Experimental breeding and genetics of mice and rats (YoSIDA and KURITA)
Biochemical study on genetical abnormalities of mice (MORIWAKI)
Determination and differentiation of sex in higher plants (TAKENAKA)
Induction of abnormal mitosis and inhibition of growth by substances
extracted from certain plants (TAKENAKA)
Interspecific hybridization in Nicotiana (TAKENAKA and FURUSATO)
Genetics of Pharbitis nil (TAKENAKA)
Origin of Prunus yedoensis (TAKENAKA)
Cytological studies on the yeast cell (YONEDA)

Department of Physiological Genetics
Genetical studies on insecticide-resistance in Drosophila (OSHIMA)
Physiological studies on eye-pigment formation in Drosophila (TAIRA and(OSHIMA)
Population genetics of deleterious genes in natural populations of Drosophila (OSHIMA)
Studies on the origin of rice (KIHARA)
Studies on nucleus substitution in wheat and related species (KIHARA)
Studies on stoneless pomegranates (KIHARA)
Monosomic analysis of 6x wheat (TSUNEWAKI)

Department of Biochemical Genetics
Biochemical genetics of insects and microorganisms (TSUJITA and NAWA)
Embryological and biochemical studies in the silkworm (TSUJITA andSAKAGUCHI)
Biochemical studies on the differentiation of muscle proteins in animals(OGAWA)
Biochemical studies on the mechanism of cell division in animals (OGAWA)
Chemical research in anti-tumor substances (OGAWA)
Biochemistry of the mechanism underlying variations in flower color inplants (ENDo)

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

Studies on fine gene structure (TSUJITA)
Genetics of virus (TSUJITA)
Immunogenetics of salmonella (IINo)

Department of Applied Genetics
Studies on breeding and genetics in poultry (YAMADA and KAWAHARA)
Quantitative genetics in Drosophila (YAMADA)
Theoretical studies on plant breeding techniques (SAKAI)Studies on competition and migration in plants and animals (SAKAI, lYAMA
and NARISE)
Population-genetic studies in' Red-Rice' growing among upland rice (IYAMA)
Genetic studies of alkaloid content in tobacco plants (SAKAI and lYAMA)
Biometrical study of sytoplasmic inheritance (SAKAI, lYAMA and NARISE)
Polyploidy and sterility in fruiting plants (FuRusATo and MIYAZAWA)
Genetic studies of some physiological and agronomic characters of rice
(OKA)

Department of Induced Mutation
Radiation genetics of mice (SUGAHARA, TUTIKAWA and MURAMATsu)
Radiation population-genetics of Drosophila (MUKAI)
Triploidy breeding of sugar beets (MATSUMURA)
Relation between the quality of radiations and mutations (MATSUMURA and
KONDO)
Radiation genetics of cereals (MATSUMURA, FUJII, NEZU and KATSUYA)
Radiation genetics and its practical application (MATSUMURA, FUJII and
MABUCHI)Radiosensitivity in plants (FUJII)
Biophysical consideration of radiation genetics (KONDO)
Radiation dosimetry (KONDO and ISHIWA)

Department of Human Genetics
Genetics of human populations (HIRAIZUMI)
Populational implications of meiotic drive with special reference to the
SD locus in D. melanogaster (HIRAIZUMI)JOINT RESEARCHES

7

JOINT RESEARCHES SUPPORTED BY A GRANT
FROM THE ROCKEFELLER FOUNDATION
Origin of Rice

1.

Section 1. Collection and preservation of Oryza species (H. KIHARA)
a. Strains of 25 species so far collected amount to more than 4,300.
b. Collection-tour was made this year by Dr. H. 1. OKA to Latin
America.
Section 2.
a.
b.
c.
d.
e.
f.

Morphology and physiology of Oryza species (S. MATSUMURA)

Comparison of radio-sensitivity of Oryza species (T. FUJII)Genome-analysis of Oryze species (H. KIHARA, S. MATSUMURA, M.
NEZU, T. KATAYAMA and T. MABUCHI)
Susceptibility of wild and cultivated rice strains to blast fungus(K. KATSUYA)
Surface structure of lemma, palea and leaves of Oryza species (H.
KIHARA and T. KATAYAMA)
Investigation of photoperiodic responses of Oryza species (T.
KATAYAMA)
Floating habit of wild and cultivated rice (H. KIHARA, T. KATAYAMA,
K. TSUNEWAKI)

Section 3.

Population-genetics in wild and cultivated rice (K. 1. SAKAI)a.

Estimation of genetic variability among and within populations ofwild rice (K. I. SAKAI, S. IYAMA and T. NARISE)
b. Estimation of the percentage of out-crossing by a biometrical method (K. I. SAKAI, S. IYAMA and T. NARISE)
c. Competition between wild and cultivated rice strains (K. 1. SAKAI
and T. NARISE)
d. Comparative studies of seedling characters of wild and cultivatoo
rice (K. 1. SAKAI, T. NARISE and S. IYAMA)e. Genetic relationship between wild and cultivated rices in Ceylon
and India (T. NARISE and K. 1. SAKAI)f. Variation studies of blast disease resistance in wild rice populations(T. NARISE and K. 1. SAKAI)

Section 4. Genetic studies in wild and cultivated rice (H. 1. OKA)
a. Statistical-systematic studies of wild and cultivated rice strains (H.
MORISHIMA and H. 1. OKA)
b. Survey of variations between O. perennis and O. sativa f. spontanea(H. MORISHIMA, W. T. CHANG and H. I. OKA)

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

c.
d.
e.
f.

Crossing-experiments and sterility of hybrids between wild and cultivated rice strains (K. HINATA and H. I. OKA)
Survey of variations between and within O. breviligulata and 0 .
glaberrima (H. MORISHIMA and H. I. OKA)
Comparative studies in O. sativa and O. glaberrima (K. HINATA, H.
MORISHIMA and H. I. OKA)
Hybrid swarms between wild and cultivated rice populations (H. I.
OKA and W. T. CHANG)

Section 5.
a.
b.
c.

Cytogenetics of Oryza species (Y. TAKENAKA)

Karyotype analysis of Oryza species (Y. YONEDA and M. SHINOHARA)
Comparative observations of chromosomes in haploid plants of Oryza
species (C. H. Hu)
Embryological studies in Oryza species (Y. DOIDA)II. Studies on genetic radiation effects on animals
Section 1.
a.

b.
c.

Two types of dose-rate dependence of radiation-induced mutation
rate observed in spermatogonia and oogonia of the silkworm (Y.
TAZIMA, S. KONDO and T. SADO)
Cytological basis of radiation-induced sterility in the male silkworm(T. SADO)
A theory for the frequency distribution of cluster mutants (S. KONDO)

Section 2.
a.

b.

Mutation in mammals (T. SUGAHARA)

Recessive lethal mutations induced by chronic irradiation given
through the whole development of mice during three successive
generations (T. SUGAHARA, K. TUTIKAWA, S. MURAMATSU and Y.
TAKADA)
Shift of secondary sex ratio among the progeny of chronically irradiated male mice (F 1 generation) (K. TUTIKAWA)

Section 3.
a.

Radiation mutagenesis in the silkworm (Y. TAZIMA)

Cytology and cancer problem (T. H. YOSIDA)

Modification of frequency of X-ray induced chromatid breaks inEhrlich tumor cells by pretreatment with DNP (T. H. YOSIDA, Y.
MATANO and K. MORIWAKI)
b. Effect of sodium azide (NaN,) on X-ray induced chromatid breaks(T. H. YOSIDA and K. UTSUMI)
c. Change of ATP content in Ehrlich tumor cells after treatment with
DNP and NaNg (K. MORIWAKI)
d. Cytological observations of ascites tumors treated with chronic rradiation (T. H. YOSIDA, T. TAKAHASHI and Y. KURITA)

JOINT RESEARCHESe.
f.

Induction of mouse leukemias by X-irradiation (T. H. YOSIDA andY. KURITA)
Chromosomes of leukemias developed spontaneously and induced by
X-rays (T. H. YOSIDA and Y. KURITA)

Section 4.
a.
b.

b.
c.

Biochemistry of radiation mutagenesis (M. TSUJITA)

The composition of ribonucleic acid in yeast irradiated with ultraviolet light (S. NAWA)
The base requirement of UV-induced mutations (S. NAKAI)

Section 5.
a.

9

Mutations in populations (C. OSHIMA)

Analysis of persistence of X-ray induced lethal chromosomes in experimental populations (C. OSHIMA and O. KITAGAWA)
Polygenic mutation rates of chaeta number in D. melanogaster (Y.
YAMADA)
Some calculations on the mutational load (M. KIMURA)FOREIGN VISITORS IN 1960
March

10.
15.
28.
29.
31.

April

May
June

July
Aug.
Sep.

5.
11.
21.
28.
29.
7.
11.
20.
1.
22.
8.
10.
12.
13.

Mr. L. F. CHAO (The Botanical Institute, Academica Sinica)
Mr. R. SHARMA (Assam Raw Silk Bureau, India)
Dr. E. E. PARCHWITZ (Atomic Energy Commission, West Germany)
Dr. J. BREWAKER (Brookhaven National Laboratory, U.S.A.)
Dr. H. KucKucK(Institute for Horticultural Plant Breeding,Hanover Technical College, Germany)
Mr. T. W. Lm (Joint Commission of Rural Reconstruction, China)
Dr. & Mrs. J. M. WILKS (Summerland B. C., Canada)
Dr. R. E. CLELAND (Indiana University, U.S.A.)
Dr. A. H. MOSEMAN (The Rockefeller Foundation)
Dr. R. A. FISHER (University of Cambridge, England)
Dr. & Mrs. H. K. NANDI (Agricultural Bureau, West Bengal,
India)
Dr. R. AUECHAGIT (Chulalongkorn University, Thailand)
Mr. G.KEMMLER, (Potash Research Association, West Germany)
Mr. J. W. FOSTER (Texas University, U.S.A.)
Dr. H. H. HUBBELL Jr. (Oak Ridge National Laboratory, U.S.A.)
Dr. H. H. CURTIS (Brookhaven National Laboratory, U.S.A.)
Dr. H. LEA (Sydney Agriculture Experiment Station)
Dr. T. MAKINODAN (Oak Ridge National Laboratory, U.S.A.)
Dr. L. C. STRONG (Roswell Park Memorial Institute, U.S.A.)

10

Sep.

Oct.

Nov.

Dec.

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

17. Mr. S. WORTMAN (International Rice Research Institute, Manila, Philippines)
Mr. P. R. JENNINGS (The Rockefeller Fundation)
26. Dr. & Mrs. F. F. HILL (The Ford Fundation, U.S.A.)
29. Mr. C. S. K. HYUN (College of Agriculture, Seoul National
University, Korea)
Mr. PHAM Huy LAN (National Agricultural Institute, Vietnam)
Mr. NGUYEN VAN CHI (National Agricultural Institute, Sough
Vietnam)
5. Dr. S. OHNO (Hope Medical Center, U.S.A.)
7. Mr. S. SAMPATH (Central Rice Research Institute, India)
26. Dr. O. MUHLBOCK & Dr. A. Dux (Holland Cancer Institute,
Holland)
7. Dr. H. LANGENDORFF & Dr. H. J. MELCHING (Freiburg University, Germany)
28. Dr. & Mrs. E. J. WELLHAUSEN (The Rockefeller Fundation)
12. Prof. G. S. STENT (California University, U.S.A.)
16. Dr. F. W. SANDERS (Oak Ridge National Laboratory, U.S.A.)RESEARCHES CARRIED OUT IN 1960
A. GENETICS, CYTOLOGY AND
BIOCHEMISTRY OF ANIMALS
1.

The persistence of induced lethal genes
in experimental populations' )(By Chozo OSHIMA and Osamu KITAGAWA)

The method of maintaining experimental populations has been describedby BUZZATI-TRAVERSO (1947). Following this method, an experimental population was started by introducing ten pairs of flies, heterozygous for an
induced recessive lethal gene, into the genetic background consisting ofinbred Samarkand chromosomes. The population has been continuously
cultured in six vials. Four populations were replicated for each kind of
lethal heterozygotes and the generations were counted in fifteen day
Table 1. Zygotic frequencies of lethal genes in experimental populations
Generationx

XIX-XXIXXVI, XXVII

0.17880.0466**0.0168**0.14820.1702** (0.0902) ,,0.0208* (0.0371),0.1234(0.0154)'0.0834(0.0816):0.0454(0.1127)0.0000** (0.0196)
0.0216(0.0901)
0.0440(0.0667)

Lethal___ ~~ne-,-s_~_-,-7
8
10
16

---

-- - - - - - , - - - - - - ----- 1 - - -mean 0.0976*,0.0995

0.24820.13800.044418
26

0.2666*---

Total mean
Theoreticalfrequency

1,-_

0 .0278(0.0766)(0.1049) I 0.0202*(0.0976)(0.0635)1(0.1081) : 0.0000** (0.0392)-------~----, mean 0.2574*

0.0912(0.1066) I 0.0101*(0.0716)0.15090.0967(0.0825)

I 0.0217(0.0750)0.1818±0.0386

0.0909±0.0287

0.0714±0.0258

(s=O)
Q2l:
Q27:

**:
1)

Expected frequency based upon the observed frequency in generation 10.
IIII

Significant at 1% level.

II

*:

II

II

II

21.

Significant at 5% level.

This work was supported by Grant RF 57178 from the Rockefeller Foundation.

12

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

periods. The number of flies in the population increased as the time
passed and reached about one thousand flies. At an arbitrarily chosen
generation, fifty male flies were sampled from two populations and they
were examined by the test-cross to the original lethal heterozygotes inorder to find out whether one of their second chromosomes had the samelethal gene or not. By means of three such sampling tests in the courseof twenty-seven generations, we could evaluate the decreasing frequenciesof the lethal gene in the experimental populations. The individual persistence of six (L 7, 8, 10, 16, 18, 26) among twenty-seven induced lethal
genes was actually followed up respectively. The zygotic frequencies ofthese lethals decreased as shown in Table 1.
The total mean zygotic frequencies of these lethals were observed todecreased from 1.000 to 0.1509, 0.0967 and 0.0219 at the initial, tenth,twentieth, and twenty-seventh generation, respectively. Four lethal heterozygotes (L 7, 8, 10, 16) out of six had relatively high coefficients of
selection (mean 5=0.18281) and the other two (L 18, 26) had negative
coefficients of selection (mean 5=-0.04055) in the homozygous genetic
background. From the results shown in Table 1, the persistence of thelatter two lethal genes during ten early generations was found to behigher than that of the former four lethal genes. However, in the later
generations all lethal genes were presumed to be similarly selected out.
As long as the genetic background of flies in the population has been kept
homozygous, lethal genes having an advantageous effect on the viabilityof heterozygotes could be allowed to persist in the population. When thegenetic background became heterozygous by spontaneous mutations in
the later generations, their superiority would be lost. The total mean
zygotic frequencies decreased showing non-significant differences from thetheoretical frequencies, when s was assumed to be zero, although theirmean 5 was 0.10836.

2.

The persistence of deleterious genes in natural populationsof Drosophila melanogaster(By Chozo OSHIMA and Osamu KITAGAWA)

The second chromosomes of D. melanogaster were isolated from several
Japanese wild populations by using the method of completely marked inversion. The relative frequencies of chromosomes carrying lethal, semilethal, subvital and normal genes were estimated. The results obtainedin 1959 had been published in the previous annual report (No. 10). A
similar sampling of second chromosomes from the same populations was

13

RESEARCHES CARRIED OUT IN 1960

carried out also in 1960 and the results were compared with those obtainedin 1959, as shown in Table 1.
Table 1.

Frequencies of deleterious and normal second chromosomes
isolated from natural populations

Percentage of wild type flies
No. of- - - ----- ---- - - - --- -- - - --. ------ - ---- - chromosomes0(1)* i 0-16.7 .16.8-25.0 25.1-33.3 33.4-41.6
tested_____ ,
(~
(sv)
(IlL
(n)
_

Population

Suyama (1959):
Suyama (1960):

9
17

6
2
-

43
84

9
18

78
133

8

23

3.79
± 1.3210.90± 2.08127
60.19± 3.3727
12.80± 2.30

211

9
3

30
34

8

56
51

I
I

---.---------

26
12.32
± 2.26 ,Total (;Yo)

1112- - - - _.. _ - - - - -JurikiJ uriki(1959)(1960)

7
6

2
1_ ... _----_ ..---Total (;Yo)

13
12.15
± 3.16 ,,* 1: lethal.

sl: semi-lethal.

--- - _ .. _ - - - -3
2.80± 1.59

12
64
59.81
11.21
,
± 3.05 ± 4.74------------------

Table 2.

7

sv: subvital.

15
14.02
± 3.36107-n:normal chromosome.

The results of allelism test between natural lethals
No. oflethals

No. ofcrossesNo. ofallelic
crossesWithin Suyama (1959)

9

Within Juriki (1959)
Between Suyama &Juriki(1959)

6

36
15

o

2.78
0.00

15

54

1

1.85

Population

Total
Within Suyama (1960)
Within Juriki(1960)
Between Suyama &Juriki(1960)
Total

IlliP~~>:i,~·1

Allelicrate-_--_-_-_-2-=-_[--;.90--_-__-__-_-_-_-_-_-_-__-~'_-__-__-__1_0_5_-_--_--_-_:-_-__
17
6

136
15

6
1

6.67

23

102

1

0.98

-

3.16

----;: -------

253

I

8

;~-----I-- ~

4.41

0.87

14

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

The different classes of deleterious chromosomes were similar in relative
frequencies in Suyama and Juriki populations and there was apparently
no fluctuation between samples collected in 1959 and 1960.
The lethal chromosomes were maintained in the Cy balanced system inthe successive generations. Diallel crosses were performed with alllethal strains to determine the allelic rate within and between populations.
After maintaining the lethal chromosomes during the year 1959, they
were subjected to cross-testing with new lethal chromosomes isolated in1960 from the same populations. The results are shown in Table 2.
The allelic rates in Suyama and Juriki populations underwent scarcely
any change during one year, but they seemed to have increased slightly
during 1960. Most interesting was that the two lethals isolated in 1959
were found again in 1960. This finding shows that the two lethals havebeen maintained in the same population at least for a year. According
to PROUT'S formula (1954), the effective size of population was deduced
from the data given in the table. The effective size of Suyama andJuriki populations was estimated to be about 1700.

3.

Heterozygous effects of natural lethal and semi-lethal
chromosomes on pre-adult viability(By Chozo OSHIMA)

The second chromosomes of Drosophila melanogaster carrying lethal,semi-lethal, subvital and normal genes were isolated individually fromseveral natural populations and their relative frequencies were determined.
The viability of heterozygotes for lethal or semi-lethal genes (or chromosomes) has been estimated by STERN et a1. (1952) and several otherinvestigators. The findings of all these authors agree in that the heterozygotes are about 3-5 per cent less viable than lethal-free zygotes.
The present author has determined the viability of thirty-five lethal
heterozygotes and thirty-two semi-lethal heterozygotes as compared withthat of normal heterozygotes carrying chromosomes free of deleterious
genes. The mean selection coefficient (5) of lethal heterozygotes wasestimated to be 0.01243 from counts of 85909 flies and the mean selection
coefficient (5) of semi-lethal heterozygotes was estimated to be 0.00678
from counts of 75482 flies.
The selection coefficients of the lethal heterozygotes were distributed
from +0.1837 to -0.1186; two of them were highly significantly lessviable than normal heterozygotes, one was significantly less viable and
one was significantly more viable, all others were not significantly differ-

RESEARCHES CARRIED OUT IN 1960

15

ent by the results of x2 -test. The analysis of variance concerning theresults of viability test was performed and on the whole no significant
differences between viabilities of lethal, semi-lethal and normal heterozygotes, were observed. The mean selection coefficient for lethal genes
in an un irradiated large population was estimated by PROUT (1954) at 0.014.
This figure is closer to ours than to those given by other investigators.
The mean selection coefficient of semi-lethal heterozygotes was about one
half of that of lethal heterozygotes. This result is different from thatrobtained by HIRAIZUMI and CROW (1960).

4.

The persistence of some natural lethal chromosomes
in experimental populations(By Chozo OSHIMA)

The several natural lethal chromosomes isolated in 1959 from the large
Kofu and Katunuma populations and also from the small Suyama andJuriki populations were introduced into individually experimental populations which have been cultured continuously in a constant-temperatureroom at 25°C. The method of maintaining these populations was thesame as described in a joint paper with induced lethal genes (Oshima andKitagawa, this Ann. Rep).
The decrease from the initial zygotic frequency 1.0000 has been followed
up in the course of fifteen generations. To determine the frequency at
an arbitrarily chosen generation, one hundred chromosomes sampled fromthe population were examined, to find out whether they have contained:fl 0 . S

"~

O.i

~

l' 0

]
~

0.5

c

... 0.4~§oO.3.:: 0.2v....................

_---------

JU1-3~

0.)Fig. 1. Persistence of lethal genes in experimental
populations.
Dotted line: theoretical curve (8=0)
L(2)55i: lethal gene found by MUKAI and BURDICK

16

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

lethal genes or not, by the test-cross to the original lethal strain. The
results are shown in Figure l.
These declining curves were compared to the theoretical curve which
was given by the no-selection coefficient (s=O). Two lethal genes, KN
1-48, KN 3-54, isolated from the Katunuma population have retained
a remarkably high frequency and most of the others have decreased ata similar rate as that of the lethal gene which showed a single gene
heterosis reported by MUKAI and BURDICK (1959).
Although these data have not been completed, they indicate that there
could have been some lethal genes involved showing heterosis in theheterozygous state in natural populations. From the results of allelism
test, lethal genes SU 3-19 and SU 2-1 were found to be identical. Thoseand SU 3-24 lethal gene were demonstrated to have been maintained, atleast for one year, in the Suyama population as described in my otherpaper.

5.

Selection experiment on migrating activityof Drosophila melanogaster(By Takashi NARISE and Kan-Ichi SAKAI)

The effect of selection on migrating activity was investigated with wildflies of D. melanogaster captured in Te-sima and Igro-sima populations,which formed the TS and IG strains, respectively. One hundred andtwenty flies (60 males and 60 females) were introduced to the" originaltube" and were kept within it for one day, then nine tubes containing
food media were connected radially to the original tube in such way thatthe flies could migrate from the original to the connected tubes throughthe connecting narrow paths. All migrants to the connected tubes werecollected for breeding the next generation. The over-all migrating activity, involving mass- and random-migrating activity, was examined inevery three generations with 30 replications. At the 21st generation of
selection, however, test was made for the random- and mass-migrating
activity separately with five replications for each. All the experiments
were conducted in a dark room at 25°C.
Variance analysis of migrating activity showed that there was a significant difference in the migrating activity among the selected lines.
Investigation of the mass- and random-migrating activity was madewith selected lines of the 21st generation of selection, some of whichbeing derived from TB and others from IG strain. In the two groups oflines, the selection for high mass-migrating activity as well as for highrandom- migrating activity was found to be quite effective. The differ-

RESEARCHES CARRIED OUT IN 1960

17

ence between the reginal unselected strains and the selected line groups
was statistically significant for both migrating activities.
Based on these results, it is concluded that migrating activity, either
random or mass, of Drosophila flies is a genetic character, and natural
populations of the fly involve a great deal of genetic diversity for thesecharacters.

6.

The effect of inbreeding on migrating activityof Drosophila melanogaster(By Takashi NARISE and Kan-Ichi SAKAI)In the preceding issue of this Annual Report, the result was describedand discussed of an experiment dealing with the mass-migrating activityof sixty-five inbred lines of Drosophila melanogaster, inbred for 20 or 30
generations. In the present report, the investigation of the same lines
with regard to the random-migrating activity is reported. Those sixty
five inbred lines were derived from four wild populations collected in 1956.
Details of the method of the experiment have repeatedly been given in
earlier papers. The experiment was replicated five times.
After 20 or 30 generations of inbreeding, those lines provided witheither very high or very low random-migrating activity were established.
It is of interest to find no apparent depression due to inbreeding in anyof the lines. That the above mentioned migrating activity might be
genetically controlled could be demonstrated by further inbreeding experiments.
Analysis between random- and mass-migrating activity showed thatthey were independent from each other, the correlation coefficient being
so low as r=O.l72.

7.

Estimation of genetic parameters in dairy cattle(By Yukio YAMADA)

Genetic parameters in the Advanced Registry Holstein cattle populationin Japan were estimated by regression method. A total of 3503 damdaughter pairs tested during the 1948~58 period were used for the analysis.
Among the total, 2401 dams were sired by 399 bulls of domestic and 652
dams 114 bulls of imported origin. Analyses were performed within these
two groups.
The average milk production in the group of domestic origin was 406 kg
less than that of foreign origin. This difference is, however, not attribu-

18

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

table to the excellency of imported bulls, because imported dam's production was 457 kg higher than that of the domestic dams. This should be
attributable to the dams' superiority owing to the breeder's choice. The
same tendency was observed in fat percentage and fat production. So, it
is safe to say that the average genetic potentiality of milk production of
the bulls of domestic origin is already on the same level as that of theimported bulls.
With respect to the variance and covariance in production traits noappreciable difference was observed between the two group, and hence
only pooled estimates of genetic parameters are given here.
Heritabilities are estimated to be 0.434±0.021 for milk yield, 0.423+0.041
for fat percentage, and 0.512±0.042 for fat production.
Genetic correlations are -0.135±0.045 between milk yield and fat percentage, 0.901±0.00S between milk yield and fat production, and 0.273+
0.05S between fat percentage and fat production.
S.

Variance increase of chaeta numbers in heterozygotes
of Drosophila melanogaster(By Yukio YAMADA)

In order to eliminate any chance of cross-over between irradiated andnormal or marked chromosomes during isogenisation processes leading to
the extraction of homozygous lines, the following simple and reliable
technique for estimating within- and between-line- variance was contrived:
In the Pi generation, a number of samples of 15 adult males from aninbred Oshyoro line were irradiated with certain doses of r-rays of 137CS
at the dose rate of 306 r/min. The irradiated 15 males of each sample
were mated immediately after the treatment to each batch of 15 virgin
females of an inbred H-SO line, whose genotype was +/+; Cy/Pm; Ubx/Sb.
Resulting F i males of genotype +/~; Pm/+; Sb/+ were selected andindividually mated to each batch of 3 virgin females of the Oshyoro line.
Scorings were made with respect to abdominal and sternopleural bristles
for 10 wild type F z individuals for each male line. The flies were h~ter0­
zygous for second and third chromosomes with respect to radiation.
Experimental results are summarized in Table l.
Increases in between-line-variance by radiation were observed and someof them reached to the significance level, while no differences among themean values were observed. The variance observed on the basis ofheterozygotes had to be doubled for comparison with random mating
condition. Thus, the increments of genetic variance give rise to thevalue of 30.94 x 1O- 5 /r and 10.0S x 10- 5/r, respectively, for abdominals andRESEARCHES CARRIED OUT IN 1960
Table 1.

Treatment

1

I

19

Means and variances in bristle numbers of heterozygotes~~:~;~~: :-M-ea-n---,A_bdO;;i~:riC~;-Me~~=~~:n~~:~:~:esamples,

Or

89
88
96
100
102
84
84

250r
500r
750 r
1000r
1500r
2000r

I

29.76
29.75

Within I Between !
5.2016: 0.6118
5.1227 I 0.6778

30.00 5.0597, 0.4820
29.45 5.4507 I 0.9191**
30.24 I 5.3769 0.8549*
30.15: 5.9114 0.6919
29.70 ! 5.4987 0.9633**

I

I

Variance increment per rad

15.47 xlO- s

I
I

!

14.68
14.73
14.32
14.99
14.87
14.48
14.65

__
Within! Between
2.7203 I 0.4764
2.62781 0.5472
2.7170 0.3683
2.78780.5181
I

2.9926 I 0.5242
2.5208 I 0.4788
2.6005 I 0.6215*

5.54xlO- s------ - - - - - - - - - -*.05<P< .10** P< .05

sternopleurals. These are considerably larger than the values obtainedon the homozygote basis as previously reported. Details will be discussed
elsewhere (Proc. Jap. Jour. Gent. 36).

9.

Heterosis and reciprocal cross differences in egg
production in some breed matings(By Takatada KAWAHARA)

During four years from 1956 to 1959, intra- and inter-breed matings,
involving White Leghorns (WL), Barred Plymouth Rocks (BPR), Nagoyas(NG) and Rhode Island Reds (RIR), were conducted. Data were collectedfrom 916 purebred and 1132 reciprocal Fl hybrid pullets. Mating systems
were such that the purebreds and crossbreds were half-sisters. Numberof eggs laid up to 120 days after the first egg was recorded for eachpullet to find hen-housed, hen-day and survivor egg production. The
means and their standard errors for the three traits obtained from pooled
four year observations, are presented in Table 1. An effect of heterosis
appeared in all traits in every crossbred. The potence ratios (MATHER
1949) of various crossbreds were 3.61, 0.93, 6.81, 10.35, 1.16, 1.62 in henhoused production, 11.68,2.78,43.90,73.32, 1.03, 1.47 in hen-day production
rate and 22.09, 5.00, 12.25, 21.72, 1.05, 1.41 in survivors production rate,
respectively, for WL-'f. X BPR-2)' , BPR-'f. xWL-2)', WL-'f. xNG-2)', NG-'f. xWL-2)',
WL-'f. X RIR -2)' and their reciprocals. The finding of differences between
reciprocal Fl hybrids in egg production performance is noteworthy. The
statistical analysis shows that the F 1 hybrids between WL sires and NG or

20

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11
Table 1.

Means of measurements for the three traits up to 120 days
after the first egg in purebred and crossbred pullets- - - -- - - - - - - - - -Hen-housed
Survivors
Hen-day' egg production I egg production egg production
rate (%)(No.)
I
rate (%)

Breed or cross------------- ---.-------- ------- -- - - - - - - - - -

WL
BPR
NG
RIR
WL+xBPR~
BPR+xWL~
WL+xNG~

NG+xWL~
WL+xRIR~
RIR+xWL~------

475
365
53
23
470
442
37
76
59
48
-- ------------

76.82±1.04
71.49±1.58
79.02±2.6250.35±4.91
83.79±1.2176.62:U .11
85.41±4.03
89.30±2.4178.97±2.46
85.06±2.34--------_..

66.16±0.77
64.66±1.1265.85±2.20
41.96±4.18
74.17±0.80
67.50±0.79
72.81±1.91
77.37±1.61
66.53±1.88
71.88±1.66

-- -

,

,

-

--

---- ----

66.92±0.84
66.11 ±1.2665.85±2.20
41.96±4.1875.46±0.83
68.54±1.06
72.94±1.94
78.00±1.35
67.50±1.83
72.00±1.82_.~._----

RIR dams produce more eggs than reciprocals. However, BPR+ x WL ~was consistently poorer in egg production performance than the reciprocal
hybrid. From these evidences either sex-linkage (WL crosses with NG or
RIR), or cytoplasm-genotype interaction and/or interaction between autosome sand sex-chromosomes (WL cross BPR), may be factors determining
the mode of inheritance for egg production.
The crossbreds appear to be less variable than the purebreds, particularly in terms of coefficients of variability. Heritability estimates forhen-housed production were 0.217, 0.429, 0.350 and 0.361 in WL, BPR.
WL+ X BPR ~ and its reciprocal, respectively.

10.

Influence of heterosis on hatchability of Fl hybrids
between two breeds of domestic fowl
(By Takatada KAWAHARA)

Within- and between-breed matings were performed using 11 sires and
84 dams of White Leghorns (WL), and 11 sires and 58 dams of Barred
Plymouth Rocks (BPR). Average coefficients of inbreeding of parental
purebreds were 2.3%, 4.6%, 5.1% and 3.9%, for WL sire, WL dam, BPR
sire and BPR dam, respectively. Mating systems were such that thesepurebreds and the Fl hybrids were maternal half-sibs, thus comparisonswere possible between groups of pure and crsssbred types. Data werecollected from 1934 purebred and 2316 crossbred fertile eggs. The average hatchability and embryonic mortality at different incubation periods

RESEARCHES CARRIED OUT IN 1960

21

and the significance tests based on the Chi-square method are given in
Tables 1 and 2.
The results of statistical analysis of the data are summarized as follows;
1) Average hatchability of fertile F, eggs was 7.4796 higher than thatof purebreds (WL=88.1696, BPR=81.4796, WL.5?xBPR~=92.5996 andBPR.5? x WL~ =91.9796), and WL was for 6.6996 higher than for BPR; thedifferences were statistically significant at the 196 level. Hatchability of
Table 1. Hatchability of fertile eggs from identical dams matedalternately with sires of different breeds,
No. of No. of No. of
I fertile
Breed or cross siresd
ams I eggs I________________,
WL
9
84
1335
WL.5? X BPR ~
9
84
1183
BPR
11
58
599
,
BPR.5? xWL~! 11
58
I
1133
,

Embryonic mortality
Hatchability-, of fertile eggs
19-22 Iin %(%1_ __

1I(incubation periods)
1-7

8-18

(d!ys)
3.75
4.40
7.68
4.59

1. 05
0.42
2 .17
0.97

_._--_ .... _ - -

7.04
2.62
8.68
2.47

88.16±2.26
92.59±2.02
81.47±4.05
91. 97 ±1. 95--------------

Table 2. Chi-square value of significance test for the differences
between breeds or crosses

Comparison

Embryonic mortality- - (inc;ibationperlods)-1-7

8-18

19-22

Fertile egg
hatched1.60
0.06
25.99**
34.29**
27.22**
32.89**

15.49**
0.29
13.78**
41. 93**
9.77**
49.33**(Iay~)

WL vs. BPR
WL.5?xBPR vs. BPR.5?xWL~
WL vs. WL.5?xBPR(1)BPR vs. BPR.5? X WL(1)WL vs. BPR.5? X WL(1)BPR vs. WL.5? X BPR(1)13.57**
0.49
0.68
7.00**
1.11
8.24**

3.78
2.53
3.29
4.13*
0.04
12.15**** Significant at the 1 % level.

* Significant at the 5% level.
WL.5? x BPR ~ was 0.6296 higher than that of its reciprocal, but thisdifference was not significant statistically.
2) Mortality of embryos after the 19th day was for F , hybrids lessthan for purebreds (WL=7.0496, BPR=8.68% vs. WL.5?XBPR~=2.6296,
BPR.5? x WL~ =2.4796), and the differences were statistically significant
at the 196 level. It is obvious that the difference in average hatchability

22

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

between the purebreds and the crossbreds was mainly due to mortality ofembryos in the late incubation period. The percentage of dead embryos
at various incubation periods were for BPR markedly higher than for theother groups.
3) Frequencies of non-genetic abnormalities observed were 0.075%,
0.334%, 0.254%, 0.618%, in WL, BPR, WL-9- x BPR ~ and the reciprocal
cross, respectively. The occurrence of abnormalities was higher among
BPR-maternal embryos than WL-maternal ones (BPR-maternal groups=
0.52% vs. WL-maternal groups=0.16%) and this difference was close to
the significance level (;(2=3.34, .1O>P>.05). The difference of 0.27% in
the occurrence of abnormalities between the purebreds and the F 1 hybrids(purebreds=0.15% vs. crossbreds=0.42%) was not statistically significant.
11.

Negative correlation between rate of development andfemale fertility in Drosophila melanogaster(By Yuichiro HIRAIZUMI)The relation between two major components of fitness, rate of development and female fertility, was studied for chromosomes II and III of
Drosophila melanogaster. The developmental rate is a major componentof pre-adult fitness and fertility is the important part of adult fitness.
The principal conclusions were as follows:
1. There was no detectable maternal effect on the components offitness.
2. To a first approximation chromosomes II and III contributed to eachcomponent in a simple multiplicative fashion although significant, butslight, deviations from this rule were observed.
3. The rate of development was negatively correlated with female
fertility when it rose above a certain level but correlation was positive
when the rate did not reach this level.
4. Some possible significance of this negative correlation in natural
populations, and the relative importance of the developmental rate indetermining the total fitness, measured in terms of Fisher's Malthusianparameter, m, were studied.

12. Some properties of the "sex-ratio" agent in Drosophila(By Bungo SAKAGUCHI and Donald F. POULSON*)

It was demonstrated by MALOGOLOWKIN, POULSON and WRIGHT (Genetics* Osborn Zoological Laboratory, Yale University, New Haven, Connecticut.
(Supported by grant 6017 from N. S. F.)

RESEARCHES CARRIED OUT IN 196023

44: 59-74, 1959) that the maternally transmitted condition known as "SexRatio" (SR) in D. willistoni can be transferred to previously normal
strains of this species. The present authors (Ann. Rep. Nat. Inst. Genetics,
Japan, 10: 27-28, 1959, Anat. Rec. 138: 381, 1960) found that the" SR "agent is present at high concentrations in the hemolymph of "SR" adultsof D. willistoni and that the agent can be transferred into D. melanogaster
where the" SR" condition has been maintained for many generations.
The nature of the" SR " agent of D. willistoni was experimentally examined from the biophysical and cytological view points.
(1) Estimation of size of the" SR" agent
In order to estimate the size of the "SR" agent, filtration experiments
were carried out by using Millipore filters with 0.3 p and 0.1 p pore size.
The supernatant obtained by centrifuging a homogenate of "SR" female
adults of D. willistoni was filtered through Millipore filters and aliquotsof the filtrates were injected into D. melanogaster hosts. The results ofthese experiments showed no reduction in infections at 0.3 p pore size and50% reduction at 0.1 p pore size in comparison with the control in which
only supernatant is injected.
The experimental determination of target size of these particles bymeans of X-ray inactivation has begun. Dosages greater than 1 x 10 5 r
result in inactivation of the" SR" properties of the particles.
(2) Assay of the "SR" agent by dilution
Inocula of differing titers were prepared by diluting hemolymph of " SR "female adults of D. willistoni with Drosophila Ringers solution. An inbred line of D. melanogaster, Oregan R, provided hosts for the titration.
Dilutions of 1: 1, 1: 10, 1: 100, 1: 1,000, 1: 10,000 were used. The incubation
period was then measured at each dilution, as were the percentage of
infections.
When a whole set of flies similarly injected is put through this procedure, some variation is observed in the individual incubation time, but
the mean is a function of the titer, being linearly related to the logarithmof the dilution. Thus multiplication of the "SR" agent follows an exponential curve similar to those for bacterial or viral multiplication.
(3) Effects of osmotic pressure on the "SR" agent
The authors (Anat. Rec. 138: 376-377, 1960) have shown by phase contrast microscopy that the blood of "SR" females of D. melanogaster, D.
willistoni, and D. nebulosa contains many minute motile granules andenormous numbers of very fine motile filaments (0.1-0.2 f.l by 4-8 p) which
possess the properties associated with small spirochetes. That these arecausally related to the "SR" condition in these species is established(Science, in press, 1961).

24

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

The effects of osmotic pressure on the fine motile filaments, in the bloodof "SR" females of D. melanogaster and D. willistoni were examined byphase contrast microscopy using various concentrations of sucrose in solution and other media. When put into distilled water, the filamentsswelled within several minutes and became spherical. Such swelling
occurs only after about 15 hours in Drosophila RINGER'S or in 0.1 M sucrose
solution. When the filaments are mixed with either 0.25 M sucrose solution or hemolymph of normal D. melanogaster, the intact form is maintained for a long time, at least for 200 hours. On the other hand, if
sucrose concentration is increased, viz. 0.4 M, 0.6 M, 0.8 M, the filaments
show shrinkage after about 70 to 100 hours.
In blood treated with penicillin the filaments become rapidly abnormal
by swelling and after one hour of treatment such blood loses the capacity
to produce "SR" infections when injected into normal hosts.
These observations support the interpretation that the" SR" agent isspirochetal rather than viral in nature in these species of Drosophila.

13.

A new case of spontaneous" post-axial polydactylism"in the house mouse(By Tosihide H. YOSIDA and Hitoshi SAKAMOTO)

Post-axial polydactylism of the front feet of the house mouse was foundby A. Nakamura in Hamamatsu Kita High School and the morphologicaland genetical characters of the abnormality were studied by the present
authors. The abnormality is characterized by the appearance of an extra
appendage on the ulnar side of one or both front feet. When only one
foot is affected it is more frequently the right one. The degree of manifestation is different by individuals, some of them having only a rudimentof a claw, but others having a claw rudiment and an abortive phalange.
Genetical analysis of polydactilism showed that this character depends ona major dominant gene and possibly some minor recessive genes which
may modify the manifestation of the character. In total of 430 miceobtained from matings between polydactyl animals for 5 generations 369(85%) were polydactyl in both front feet, 35 (8.1%) in the right foot only,
and 5 (1.1%) in the left foot only. The remaining 21 mice (5.8%) had
normal feet. In outcrossing to various inbred strains (C57BR, DBA/Ma,
dba and C3H) polydactilism was found in about one half of the F,. In
F 1 the frequency of polydactyl females amounted to 60.3% while in thereciprocal cross it was only 42%. Female mice seem to be somewhat
more liable to the manifestation of polydactylism than the males.
F 2 offspring from crosses between F 1 and normal mice obtained from

RESEARCHES CARRIED OUT IN 1960

25

C57BR (¥) X polydactyl (~\ developed the charactor in 10.4%, while thosefrom polydactyl (¥) x C57BR (I)) were observed to show polydactylism in
33%. Based on the above investigation it is assumed that the manifestation of polydactylism may be affected by the cytoplasm of the mother.

14.

Invasion of tetraploid cells from a common strainof Yoshida sarcoma(By Tosihide H. YOSIDA)

Last year, the invasive ability of diploid and tetraploid tumor cells wasstudied with a subline of YOSHIDA sarcoma in which tetraploid cells occurred at a high percentage (YOSIDA 1960). Intraperitoneal transplantationof lung tissue of the tumor bearing rat resulted in a remarkable increaseof tetraploid cells. It was concluded that tetraploid tumor cells invaded
the organs easier than diploid cells.
In order to reinvestigate the infiltration ability of tetraploid tumor cells,
a common strain of YOSHIDA sarcoma was used. This tumor contained
tetraploid cells at 7.3%. Their number was reduced to 7.0% by intraperitoneal transplantation of ascites tumor, while it was increased to 12.0 and
33.3% by intraperitoneal inoculation of spleen tissue of the same tumor
bearing animal. When kidney tissue was inoculated the frequency oftetraploid cells increased to 19.0%, while it decreased to 2.5 and 5.3% in
two rats inoculated with thymus tissue of the same rat. By serial transplantation of spleen of two rats in which tetraploid cells occurred at
33.3%, the frequency of tetraploid cells increased markedly to 50.6% in one
and 68.0% in the other rat. The results indicate that the infiltration
ability of tetraploid YOSHIDA sarcoma cells into lung, spleen and kidney
was much stronger than that of the diploid stem cells, while the latter
showed a predominant proliferation in the thymus gland.

15.

Invasiveness of hypertetraploid tumor cells from a hyperdiploidstrain of Ehrlich ascites carcinoma(By Tosihide H. YOSIDA)

As shown in the preceding report, the tetraploid cells of YOSHIDA rat
sarcoma had stronger infiltration ability into some organs than diploidcells. By double inoculation with hypotetraploid (EL T) and hyperdiploidcell strains (ELD) of Ehrlich ascites carcinoma, YOSIDA and lTOH (1960)
found that the ELD tumor cells were much stronger in competition thanthe ELD cells in the case of ascites tumor inoculation, but the latter

26

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

showed a higher ability than the former to invade a few organs. In order
to investigate in detail the invasive ability of tetraploid cells, the ELD
strain was used. This tumor contained hypertetraploid cells at 13.0%.
After the us.ual transplantation of the ascites tumor the frequency ofhypertetraploid cells decreased slightly to 11.0% at the next transplant
generation, while it increased to 71.0 and 30.9% in two mice which were
inoculated with lung tissue of the same tumor bearing animal. By intraperitoneal transplantation to two mice of the ascites tumor of the former
mouse which had developed by lung tissue inoculation, the frequency ofhypertetraploid cells decreased to 67.0% in one and 46.2% in the other.
Frequency of hypertetraploid and hyperdiploid cells in the metastatic
tumors. of ELD strain was investigated by intravenous injection. This
strain was characterized by containing only 0.2% hypertetraploid cells.
By intravenous injection of the tumor cells into tail veins two micedeveloped tumors. One of them developed a tumor under the skin in theupper part of the ventral side and the other developed two tumors on thedorsal side and in the kidney. These three tumors were separately injected into peritoneal cavities of normal mice. Four mice inoculated withthe ventral tumor pulps developed ascites tumors. They contained tetraploid cells at 85.0, 47.3, 57.0 and 57.8%, respectively. One ascites tumor
developed by inoculation of the dorsal tumor was characterized by tetraploid cells at 40.4%, while the transfer of tumor pulps developed in thekidney resulted in the development of two ascites tumors. They contained tetraploid cells at 22.6 and 11.6%, respectively.
Based on the above investigations the conclusion may be drawn thatthe hypertetraploid tumor cells which were contained in the ELD strain
had a higher ability for invasion or metastatis.

16. Chromosome condition of hypertriploid Hirosaki sarcome(By Kazuei TANAKA and Tosihide H. YOSIDA)

USUBUCHI and ABE (1960) established a tetraploid subline of the Hirosaki
sarcoma and reported that the modal occurrence of the chromosome
numbers in its cells was 70-72. The present authors reinvestigated the
karyotypes of 68 cells of the subline. According to our investigation, thenumber of chromosomes varied from 54 to 109 with the mode at 67(29.4%) The number of rod-, V- and J-shaped elements was also variable,
and the mean frequency of their occurrence was 25.66+1.05, 18.44±0.92
and 23.04±0.94, respectively. Numerical variation of the characteristic large
V-elements from 2 to 6 was observed, showing the model occurrence at
4. However, there was no correlation between the total chromosome

RESEARCHES CARRIED OUT IN 1960

27

number and the number of V-shaped elements.
Based on the above results it is assumed that the cells with hypertriploid chromosome constitution may be in the process of transformationof the tumor stem line.

17.

Alteration of cell populations in serial transplantationsof hypotetraploid Ehrlich carcinoma(By Yoshikazu MATANO)

The hypotetraploid Ehrlich carcinoma was obtained from the Institutefor Infectious Diseases, Tokyo, through the Zoological Institute, Universityof Tokyo, in March, 1960. Since then this carcinoma has undergone 41
transplantations in Swiss albino mice inbred in our institute. In the earlysamplings the carcinoma was characterized by hypotetraploid 75 chromosome stem cells with two large metacentric marker chromosomes.
Recent samplings (December, 1960) from the 31st transfer generation
revealed, however, that the stem-cells of this carcinoma showed the addition of one outstandingly large telocentric chromosome. In order to findits origin, the preparations from the 11th transfer generation were reexamined with the result that the cells with the outstandingly large telocentric chromosome already existed in the tumor population though invery low frequency.
Previously, it was reported by YOSIDA (1959) that in a new subline of
Yosida sarcoma two types of stem-cells existed together for about 20
transfer generations. A similar observation was made in MTK-sarcoma
II by the present author (MATANO 1960). In the present case found in the
Ehrlich carcinoma a gradual alteration of stemline chromosomes occurredin the course of transfer generation.

18. Proliferation of Ehrlich ascites carcinoma cells in vitro(By Kazuhiko R. UTSUMI and Tosihide H. YOSIDA)

Hyperdiploid Ehrlich ascited carcinoma cells were withdrawn by a syringeon the seventh day after transplantation from peritoneal cavities, washedwith a sufficient volume of Hanks' solution, then centrifuged for 10
minutes at 1,000 rpm. The cellular sediment was resuspended in a partly
synthetic culture medium of the same volume. The cells in the suspension amounted to several ten millions per milliliter. This suspension wasplaced in flat test tubes or TD-flasks and incubated at 37-38°C. The
medium was renewed every other day during two weeks and thereafter
every 3-4 days.

28

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

Observation was made in situ every day with an inverted phase-contrast
microscope. After a few hours, the cells became attached in a mass tothe bottom of flasks and almost all died within a week. They were notremoved until subcultures were made, since a few surviving cells wereamong them which could be selected for further subcultures. After twoor three weeks, clonal divisions appeared here and there and later, the glass
surface was replaced by a living cellular monolayer. Subcultures from theprimary culture were made weekly, and after three or four weeks the cells
began to show a striking growth, and assuming spindle or polygonal shapes,
they migrated to the glass surface. The growing cells were flat or round;the former were living sound cells which contained large nucleoli andappeared to have cellular active metabolism, while most of the latter wereconsidered as dead. In the flasks, small amounts of cells or cell-clumps
were usually detached from the monolayer and were suspended in themedium. When they were placed in new flasks, they could proliferate on
the glass surface as before. This indicates that some of the round cells
were alive.
Thereafter the subcultures were made successively every week or every
ten days. Two separate attempts at adaptation in vitro of the ascites
cells gave similar results, and it may be assumed that the adaptations
begins after a month, or in other words the cell population which can be
subcultured needs at least one month to establish itself, and thereafter
subcultures may be successful. In both cases, cells were maintained for
4-5 months, with three transplant-generations in one case and ten in another case. The results indicate that Ehrlich ascites carcinoma cells
require merely a long term nourishment for adaptation to in vitro conditions.

19. Studies on mouse leukemia I. Incidence of leukemia in
AKR/fax strain mice bred in Misima(By Tosihide H. YOSIDA, Yoshinori KURITA and Hitoshi SAKAMOTO)

AKR/Jax strain mice were obtained from the Jackson Memorial Laboratory
by the senior author in 1958, and were later bred in his laboratory. The
incidence of spontaneous leukemia in this strain was 89.39%. In males
it amounted to 87.9% and in females to 90.6%, with no significant difference between the two sexes. The analysis of the leukemias showedthat the percentage of generalized lymphatic leukemias was in females
27.6%, and in males 24.1%, and that of thymic lymphosarcoma was in
the former 25.9% and in the latter 22.41%.
A comparison of blood counts of leukemic and non-leukemic mice showed

RESEARCHES CARRIED OUT IN 1960

29

-that the ratio of red blood cell counts for these two groups was (381.9±17.6)x10 4 : (940+72.0) x 10 4 and that of white blood cell counts (38.19+32.77)x10 3 : (1O.82±1.44) x 10 3 • The ratio of young forms of neutrophiles
was for leukemic and non-leukemic mice 3.33: 0.06; of segment forms ofneutrophiles 5.67: 34.25; of eosinophiles 0: 2.38; of monocytes 1.6: 2.38;
and of lymphocytes 89.43: 61.44.
Regression of age on the incidence of leukemia was linear. Regression
line is shown as Y=12.11X-69.92, where Y stands for the leukemia
incidence and X for the age in months beginning with the sixth month.
The t-test of the regression coefficient showed that it was highly significant (P<O.OOl).
Though the incidence in females did not differ significantly from thatin the males, the regression coefficients were significantly different fromeach other. In females the incidence was Y=12.41X~73.43, and inmales, Y=7.73X-26.31.
Based on the above investigation it is assumed that males of the
AKR/JaxMs strain have slightly stronger resistance to the developmentof spontaneous leukemia than the females. It seems that sex hormones
affect the incidence of leukemia in this strain. Usually, leukemias inmale mice of AKR/JaxMs strain developed at a considerably older age
than in the females; the average age of females developing leukemia was
10.25 months but 12.25 months in males.

20.

Studies on mouse leukemia II. Susceptibility to radiation induced
leukemias in various substrains of mice bred in Misima(By Yosinori KURITA and Tosihide H. YOSIDA)

The total number of 238 mice covering 8 substrains (C3H/HeMs,
C57BL/6Ms, C58/Ms, AjHeMs, DM/Ms, C57L/Ms, RF/Ms and SLIMs) wereused for induction of leukemias by X-irradiation. X-rays were given to
the total body at the average age of 2.0 months. The conditions were as
follows; 250 kVp; 1.5 rnA; 0.3 mm Cu+0.5 mm Al filter; TDS 50 cm;
60.8 r/min. Total 600 roentgen were given in four 150 r fractions at one
week interval. The mice were kept about 2 years.
Leukemia incidence in C57BL/6Ms strain was 21.43 percent, in RF/Ms
strain 71.5 percent and in C58/Ms strain 10.0 percent. The average age
before leukemia development was 10.5 months in C57BL/6 strain, 4.5
months in RF/Ms strain and 12.0 months in C58/Ms strain. In RF strain
0.03 mg of 20-methycholanthrene was injected subcutaneously after total
300 r irradiation.
Although C58 strain mice are known as a high leukemic strain, no

30

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

mice of this strain in our colony had developed spontaneous leukemia in
the past 8 years, but in the above experiments a relatively high susceptibility to radiation induced leukemia was found in this strain. In RF
strain mice leukemia induction was accelerated if they were irradiated in
combination with application 20-methylchrolanthrene.
Leukemias developed in C57BL strain had the character of thymic
lymphosarcoma, while in RF strain they had the character of generalized
lymphatic leukemia; myeloid leukemias were never seen in this strain.

21.

Studies on mouse leukemia III. Chromosomes in spontaneousand induced mouse leukemias(By Yoshinori KURITA and Tosihide H. YOSIDA)

Chromosomes in nine spontaneously developed leukemias in AKR strain
were analysed. Among them five (AKLA, AKLD, AKLE, AKLF and
AKLJ) were characterized by the model chromosome number 40 and two(AKLC and ALKH) had the mode at 41 chromosomes. Of the remaining
two one (AKLI) had bimodal distribution with one mode at 40 and theother at 41 chromosomes and the other (ALKG) had the mode at 40 andhypotetraploid numbers (70-80). All chromosomes were characterized byrod shape like those of normal somatic cells. Among leukemias inducedby X-irradiation 3 strains (BLXLA, RFXLB and RFXLC) were observed.
The mode in BLXLA tumor cells was 40 and they were characterized by
the normal rod shape. It is noteworthy that 21% of cells had 41 chromosomes in 1-3 transplant generations, which were reduced to 3.0% in the
26th transplant generation, whereas the number of 40 chromosome cells
increased to 63%. No chromosomal differences were found between spontaneous and induced leukemias. Among two leukemias developed in RFstrain, one (RFXLC) was characterized by the modal chromosome number
at 41, but the other (RFXLB) was characterized by bimodal occurrence of
41 and 42 chromosomes.

22. Susceptibility of rats to tumor induction
by treatment with methylcholanthren(By Tosihide H. YOSIDA and Yoshinori KURITA)
Wistar/Ms and Long Evans/Ms rat strains were used for the test ofsusceptibility to 20-methylcholanthren-induced tumors. Total number ofanimals used in the present study was 45 (WISTAR, 26, and LONG EVANS,.
19). All of them were about 2.5 months old. 0.5 mg of 20-methyl-RESEARCHES CARRIED OUT IN 196031,.colanthren suspended in propylen glycol was injected under the skin on
the ventral side of each animal.
The incidence of tumors developed after injection of the chemical wasin Wistar rats 71.4% and 85.7% in Long-Evans rats. Animals in bothstrains developed tumors 4.5 months on an average after treatment withthe chemical. There was no difference between the strains in the latent
period preceding the appearance of tumors. Neither there was a significant difference in the incidence of tumors between both strains (X 2 =0.654,
0.30<P<0.50) or both sexes (X 2 =0.701, 0.3<p<0.5 for Wistar strain rats,
and X2 =1.296, 0.20<p<0.30 for Long Evans strain).
Transplantation experiments with these tumors were all negative; thetumor pulps were injected subcutaneously to 30 young animals of eachstrain, but no growth of grafted tumors resulted. A cytological study ofthese tumorsris now in progress.

23.

]v[afernal effect of +lem gene on pterine reductase(By Mitsuo TSUJITA)

Recently, the structure of the yellow pigment in the silkworm wasstudied by NAWA who ascertained that this pigment is 2 amino-4 hydroxy-6
lactyl-7·8 dihydropteridin. This revealed that the yellow silkworm pigment is the same as sepiapterin of Drosophila melanogasfer. A study on
pterine reductase and on the maternal effect of +lem gene on this enzyme
was carried out. The outline of the experimental results is as follows:
The activity of pterine reductase was observed in the integument, adipose tissues, mid-gut and silk-gland of larvae, but not in their body fluid.
In the pupal and adult stage, the enzyme activity was detected not onlyin the integument, adipose tissue, and male and female genital organs
including testes and ovarian tubules but also in the bady fluid. Among
various tissues adipose tissue showed the strongest enzyme action.
On the contrary, enzyme activity could not be detected in several tissues of larva, pupa and adult lemon. However, as adipose tissue andintegument exhibited a weak or very weak enzyme activity, it seems thata small amount of the enzyme can be produced in some tissue cells of
the lemon individuals. But in the integument and in several tissues ofthe lethal lemon larvae immediately after the first moulting no enzyme
activity could be detected. Therefore, it appears that in this mutant it
is almost completely lacking.
From these experimental results it may be safely concluded that the accumulation of the yellow pigment in the hypodermis of lemon and lethallemon larvae is due to low activity or lack of enzyme action controlled

32

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

by +lem gene.
Eggs immediately after having been laid by normal moths show enzyme
action although it is weaker than in the maternal body before egg laying,
The enzyme activity of the eggs becomes stronger one day after they
were laid, and this condition continues for several days; further stronger
activity was observed with approaching hatching. On the contrary, inthe lemon eggs (lem/lem, lem/lem 1) enzyme activity was scarcely recognizedfrom the stage immediately after they were laid until the stage of
hatching.
In the eggs of the cross + x lem, almost the same enzyme activity as
in a normal strain can be observed. In the eggs of the cross lem x +,enzyme activity is scarcely observed for 30~40 hours after they were laidat the room temperature of 23~25C, but it appears thereafter. Although
the activity increases with incubation, it is always weaker than in theeggs of the cross + x lem. The same condition was observed even at
the stage of young larvae immediately after hatching.
Almost the same maternal effect of +lem on pterine reductase can beobserved in the reciprocal crosses between +/lem 1 and lem/lem 1.
Thus, a clear-cut maternal effect of +lem on pterine reductase has been
proved.
In another experiment, mutual transplantation of ovaries between normal and lemon larvae was carried out and the enzyme activity of theovarian tubules which developed in the body of the female host wasmeasured. It may be concluded from the result of this experiment thatthe enzyme activity of the eggs is not determined by their genotype butby that of the maternal body in which the eggs develop. It is likely thatthe enzyme or it's components may be transmitted from mother's tissues
to the developing egg cells in the transplanted ovaries through body
fluids.

24.

Genetical and biochemical studies on the metabolism of
pteridines in silkworm- The structure of the yellow pigment(By Saburo NAWA)

It was established that the yellow pigment occurring in the epidermisof the mutant lemon has the structure 2-amino-4-hydroxy-7,8-dihydro-6lactylpteridine, which is identical with the structure of Sepiapterin foundin Drosophila. The experiments were carried out in a similar way as
those in Drosophila (see following papers: This Annual Report, 10 104-105(1959) and Bull. Chem. Soc. Japan, 33 (10), 1555-1560 (1960)).

RESEARCHES CARRIED OUT IN 1960

25.

33

Biochemical studies on drosophila pterine reductase(By Toshifumi TAIRA and Saburo NAWA)

It is a well-known fact that the red and yellow eye-pigments, namely
drosopterin and sepiapterin, found in D. melanogaster are the products ofpteridine metabolism. For analysis of the mechanisms of the eye-pigment
formation in vivo, therefore, it is highly important to clarify in detail thepteridine metabolism.
Pterine reductase extracted from Drosophila converted sepiapterin into
tetrahydro-form in the presence of reduced triphosphopyridine nucleotide(TPNH), as TAIRA (1960) has reported. The reductase has been further
purified and its properties have been examined. An enzymatic reductionof sepiapterin could be detected spectrophotometrically by disappearanceof the yellow color and fluorescence characteristic of sepiapterin. The
curve of product converted enzymatically was similar to those of tetrahydropteridines reported by several authors. Stoichiometrical analysis
showed that one mole of TPNH was necessary for one mole of sepiapterin
reduced. These findings suggest that the tetrahydro-form converted
enzymatically from sepiapterin is tetrahydro-sepiapterin and that the present enzyme is a true reductase.
In the absence of oxygen, the tetrahydrosepiapterin converted enzymatically from sepiapterin was gradually converted further products by nonenzymatic oxidation. In this case, the main product was indistinguishable
from biopterin in various R f values and chemical natures examined.
In the presence of oxygen, the tetrahydrosepiapterin was oxidized into
two kinds of products; one of them was in a trace of original sepiapterin,
and the other was in a great amount of blue fluorescent pteridine. The
latter consisted of further two kinds of pteridines; one was 2-amino-4hydroxypteridine and other 2-amino-4-hydroxypteridine-6-carboxylic acid.
On the basis of these experimental results, the metabolic relationships
among drosophila pteridine derivatives could be presented as follows:

Sepiapterin ~ Tetrahydrosepiapterin ~ [Dihydrobiopterin] ~ Biopterin~

2-amino-4-hydroxypteridine
It remains elucidate, however, what is the immediate precursor of
drosopterin and what mechanism underlies the process of its formation.
According to our previous reports, it is assumed on the basis of geneticalanalyses that drosopterin might be converted from sepiapterin. The involved enzymatic system is controlled by the mutant gene Hn r3 • The
enzyme activity of a Hn r3 male was much lower than that of a normal male.
This may be due to a high concentration of an inhibitor, which is a deriva-

34

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

tive of pteridines. The detailed results of the continued investigation will
be published elsewhere.

26.

Phosphatase activities in the skin of hairless mice mutants(By Kazuo MORIWAKI)

As shown in FRASER'S experiments (1946), the hairless character of therhino mouse is fixed in the skin immediately after birth and becomes apparent 2 weeks later. As a preliminary step to a study of the developmental process leading to hairlessness, the biochemical changes accompanying the manifestation of the character were examined.
In rhino mice (hr rh ), acid phosphatase (Pase) activity in the skin of thehomozygotes was markedly higher than that of the heterozygotes as previously described (MORIWAKI, 1959). This result suggested a comparison
between homo- and heterozygotes in the activities of inorganic pyrophosphatase (PPase) and apyrase. A considerable increase in PPase activity
could be observed in the homozygotes in parallel with depilation, whereas
no notable change in apyrase activity occurred. After depilation, theglycogen content was markedly increased in the homozygotes, in which
an appreciable increase in lipid phosphate content was also found. Effectsof metals On acid Pase activity both of homo- and heterozygotes could be
hardly recognized using Mg++, Ca++, Fe++, Zn++, Sn++ and EDTA at concentrations from 1O-2M to 1O-4M. To confirm the relationship between
hairlessness and the above chemical changes other hairless mutant mice
were employed.
Similar results as those obtained with rhino mice have been obtained withalopecia mice (ap) which are characterized by cyclic monthly depilation. It isnoteworthy that in this case reproduction of hair in the skin of homozygotes
was also accompanied by a considerable decrease in acid Pase activity.
Furthermore, in another hairless mutant strain, furless Us), the relationship between hairless and acid Pase activity increase was nearly the~same; i.e. acid Pase activity in the homozygotes was increased by about
40 per cent over that in the heterozygotes.

27.

Comparison of the DNA content of diploid and tetraploid
Ehrlich ascites carcinoma cells according tomicrospectrophotometer measurements(By Kazuhiko R. UTSUMI and Tosihide H. YOSIDA)

The relation between DNA-content and chromosome number was studied
in the diploid (ELD) and the tetraploid strain (ELT) of the Ehrlich ascites{carcinoma cells. The DNA measurements were made with a microspec-

RESEARCHES CARRIED OUT IN 1960

35

trophotometer (Olympus) adopting the two-wavelength method.
On the seventh day after transplantation of the Ehrlich ascites tumor,a high rate of growth and abundant mitoses were observed. The ascites
exudates were withdrawn with a glass capillary from the peritoneal cavity
of a tumor bearing mouse. One part of the sample was used for DNA
measurements and the other part for the analysis of the chromosome
numbers.
Chromosome numbers: From squash preparations of both strains of the
carcinoma, stained by acetic dahlia, a hundred well spread metaphases
were examined. The mean chromosome number was 44.51 for the diploidcells and 73.49 for the tetraploid cells. Thus, the former strain was hyperdiploid and the latter hypo-tetraploid.
DNA measurements: The tumor exudates were smeared on slides, fixedwith Carnoy's solution for 20 minutes. The smear preparations werehydrolysed for 10 minutes with IN-HCI at 60-+0.5°C, then stained for
1.5 hours with Schiff's reagent at room temperature, and later bleachedwith glycine-buffered SOz-water for 15 minutes. The relative amounts of
DNA were measured by a microspectrophotometer according to Patau's
formula using the wavelengths of 482 m,u and 561 m,u. One hundred interphase nuclei of ELD, and 200 interphase nuclei of ELT cells were usedfor DNA measurements. The mean amount of DNA was 0.093 for ELD
and 0.136 for ELT. Further, the DNA-contents of resting nuclei of normal somatic cells of mouse, monocytes from ascites fluid and splenic cells
were also measured. The mean DNA-content of somatic nuclei whose
chromosome number was reported as 40 by MAKINO was 0.080. In comparing the observed mean values of DNA-content with the expected values.
computed from the mean chromosome numbers, no significant difference
was found between them.
From those results it is concluded that DNA measurements could be
used for the determination of ploidy of resting nuclei.
28.

DNA-content of Ehrlich ascites carcinoma cells withspecial reference to its variation during mitosis(By Kazuhiko R. UTSUMI)The DNA measurements were made with a microspectrophotometer(Olympus) adopting the two wavelength method. The relative amounts of
DNA were computated according to Patau's formula. The two wavelengths employed were 482 m,u and 561 m,u.
The hyper-diploid strain (ELD) and the hypo-tetraploid strain (ELT) of
the Ehrlich ascites carcinoma cells were employed. On the seventh day

36

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

after transplantation when the tumors showed a high rate of growth andabundant mitoses, ascites exudates were withdrawn with a glass capillary
from the peritoneal cavity of a tumor bearing mouse. The tumor samples
were smeared on slides, then fixed with Carnoy's solution. Hydrolysis
with 1N-HCI was applied for 10 minutes at 60+0.5°C. After hydrolysis,
the smear preparations were stained with Schiff's reagent for 1.5 hours
at room temperature. Thereafter, they were bleached with glycinebufferedSOz-water for 15 minutes.
DNA-content of nuclei at various mitotic phases: The amounts of DNA
of nuclei were measured at interphase, prophase, metaphase, anaphaseand telophase of both cell strains, hyper-diploid and hypo-tetraploid. In
both the change in DNA-content of nuclei during mitosis took a parallelcourse. The amounts of DNA at metaphase, were twice as much as thoseat anaphase and telophase. On the other hand, the amounts of DNA at
prophase were approximately 45% of those at metaphase. In interphasenuclei of ELT the distribution of DNA amount from nucleus to nucleus
was bimodal. Two groups could be established: nuclei containing lower
amounts (lower groups) and those containing higher amounts of DNA(higher group). The amounts of DNA of the lower group were consistent
with those of ana- or telophase nuclei, while the amounts of DNA of thehigher group were consistent with those of prophase nuclei. The same
analysis was made of ELD cells, which showed a unimodal distribution,and the same results were obtained.
Nucleus size and DNA-content: Further, the correlation between the
DNA-content and size of nuclei was investigated. The diameter of nuclei
at interphase and prophase was measured with an ocular sliding-micrometer and the data were used for computing their area. Since the nuclei
were flattened by being dried on the slide and had a discoidal shape, theirarea served for comparing their relative size. There was a close correlation between the DNA-content of nuclei and their size. In general, thelarger the size, the higher was the amount of DNA. The dispersions ofprophase nuclei corresponded with those of the higher group in bothstrains of carcinoma cells.
These results support the view that doubling of DNA content occurs
during interphase in agreement with the data of SWIFT (1950), FOWARD and
PELC (1952), KIMBALL and BARKA (1959).

RESEARCHES CARRIED OUT IN 1960

29.

Two factors influencing sensitivity to x-rays in the synthesisof actin during early embryonal stages(By Yoshito OGAWA)

In a previous note (Y. OGAWA, Nature. 186, 77. 1960) it was shown thatthe synthesis of muscle proteins takes place in early Triturus embryos
irradiated by x-rays (50 r, 200 rand 500 r) 108 hrs. after fertilization. On
the basis of this finding an investigation of the mechanism of thesynthesis of muscle proteins in early developmental stages seemed to bepromising. Therefore, actin formation with respect to age sensitivity inrelation to x-rays as found in the embryos of Triturus pyrrhogaster, Boie
has been studied in detail and the results are here reported.
Approximately 7,500 embryos at different developmental stages were
exposed to x-rays at single treatments with 5 r, 10 r, 30 r, 50 r, 200 r, 500 r,
1,000 rand 2,500 r. Examination for the first detectable trace of actin
after fertilization was carried out in periods of 24 hrs. after irradiation.
The technique employed was essentially the same as before (Y. OGAWA,
Nature. 182, 1312. 1958). G-actin, prepared from adult Triturus, was injected into rabbits and the obtained anti-serum was raised to their serological specificity by resorption test with saline extracts of liver, spleenand skin tissue of Triturus. The titre of antiserum was adjusted to
1 : 512 before carrying out the precipitin reaction with saline extracts of
x-irradiated embryos.
In normal embryos, actin first became detectable 132 hrs. after fertilization. The time of actin formation after fertilization in the treated embryos
is shown in Fig. 1. A dose of 5 r given 36 hrs. after fertilization shows

1~1
156

1

j 132f

i
"§

108

1

r

; :t

I

1
36 •

I

I

200r

L~~

30r

lOr

5r

SOr

~.2,500r~ ·~-~108'--'1~56--"2",o.---':25"'2 - - Age at actin ronnation (Hours)

Fig. 1. Synthesis of actin in early Triturus embryos
irradiated by x-rays applying doses from 5 r to
2,500 r.

38

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

no effect on actin formation (Fig. 1*), but in the case of irradiation 108
hrs. after fertilization with a heavy dose of 2,500 r, first actin formation
was recognized 252 hrs. after fertilization (Fig. 1**).
The curves shown in Fig. 1 indicate the presence of two factors influencing sensitivity to x-rays in the synthesis of actin during early embryonal stages. One (A) promotes the synthesis of actin and the other(B) markedly suppresses it after x-irradiation. Factor A is very sensitive
to x-rays and is easily detectable with only 5 r doses. On the contrary,factor B does not react to x-rays until doses over 50 r are applied. Factor
A is clearly detectable by irradiations between 36 and 84 hrs. and themost effective time of irradiation in the above experimental conditions is
60 hrs. after fertilization. Exposures between 84 and 108 hrs. after fertilization show the reaction of factor B to x-rays. The most effective timeafter fertilization of x-irradiation for factor B is 108 hrs.
The differentiation of muscle tissue is not asccomplished before bothactin and myosin formation coincide. Studies on myosin formation in xirradiated Triturus embryos are on the way.

30.

Biochemical relation between the syntheses of actin andmyosin in the regenerating limb tissue of Triturus(By Yoshito OGAWA)

In the skeletal mascle the formation of actin precedes that of myosinin the early developmental stages as well as in regenerating tissues.
The following questions therefore arose: Does the formation of actin
induce that of myosin? According to my previous note, it had been already proved by the investigation of the synthesis of both those proteinsin the X-irradiated embryo that during the skeletal muscle differentiationin the early developmental stage the formation of actin does not inducethat of myosin. The present note deals, from the same view point, withthe synthesis of muscle protein in the regenerating hind-limb tissue after
amputation at the knee of adult Triturus pyrrhogaster. The same serological technique was used as that employed in the experiment with early
embryonal stages.
G-actin and myosin, prepared from the skeletal muscle of adult Trituruspyrrhogaster by the method of Straub and Szent-Gyorgyi, were injected
into rabbits and the obtained anti-sera were raised to their serological
specificity by a resorption test with saline extracts of liver, spleen andskin tissue. The titres of both sera were adjusted to 1 : 512 before carrying out the precipitation reaction with salin extracts of granulated tissue of the amputated limbs. Total body x-irradiation with 200 r, 500 r,

RESEARCHES CARRIED OUT IN 1960

39

1,000 rand 2,500 r was made 18 day after the operation.
In the non-irradiated control, actin and myosin first became detectablein the regenerating limb tissue 20 and 27 days after the operation, respectively. (Y. OGAWA, Nature. 182, 1312. 1958).
In the case of irradiation, the time of myosin formation after operation
was changed to 24 days with 200 r, 22 days with 500 rand 20 days with1,000 r, though no significant influence was found in actin formation withthe above mentioned x-ray doses. (Fig. 1). A heavy dose of 2,500 r changes

I

2~r

"

~

I

81~r

1~__~_,--,;,;_~_-=_M-,Y"";;-'_-.._
£v

22

24

Z6

28

30

Days after operation

Fig. 1. Synthesis of contractile proteins in regenerating limb tissue of Triturus pyrrhogaster irradiatedby x-rays 18 days after operation.

the time of actin and myosin formation to 26 and 22 days after operation, respectively. Thus, x-irradiation 18 days after the operation, markedly
promoted the synthesis of myosin and suppresed the synthesis of actin.
The order of actin and myosin formation in the regenerating limbs tissueof irradiated Triturus at the heavy dose of 2,500 r was reversed to thatfound in the non-irradiated control. This finding is in full agreementwith the results of the experiment with early embryonal stages.
Accordingly, it may be concluded that in the skeletal muscle theprocesses of synthesis of those two proteins are independent of each otherin the regenerating tissue as well as in the early developmental stages ofthe embryo.

40

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

31.

Effects of glucuronic acid and related compounds on the synthesisof skeletal muscle proteins in early Triturus embryolJ(By Yoshito OGAWA and Noriko KARUBE)

This note deals with the effects of Na-Glucuronate, glucosamine andglucuronolactone on the synthesis of skeletal muscle proteins in early
Triturus embryos.
Immediately after fertilization, the embryos were raised in 0.01% solution of the above three chemicals at 20°C and actin and myosin formationin the developing embryos was investigated. The identification of muscle
proteins was carried out by means of a serological technique (Y. OGAWA,
Nature. 182, 1312. 1958).
In the non-treated control group, actin and myosin first became detectable in early embryos 132 and 176 hrs. after fertilization, respectively.
Na-Glucuronate had no remarkable influence on the formation of muscle
proteins, but glucosamine promoted the synthesis of myosin only and bothproteins were detected in embryos treated with glucosamine 132 hrs. after
fertilization. In the embryos treated with glucuronolacton, actin andmyosin were first detected after 156 and 108 hrs. respectively. Thus,
glucuronolactone suppressed markedly the synthesis of actin and promotedthat of myosin. The order of actin and myosin formation in embryos
treated with glucuronolacton was therefore reversed to that found in
the non-treated control.
The three compounds have the same main chemical structure and are
different only in the free radical. The above results may be useful toresearchers of the mechanism of muscle protein synthesis during early
embryonal stages.

B.

GENETICS, CYTOLOGY AND BIOCHEMISTRYOF PLANTS
32.

Genome analysis in the genus Oryza II2)

(By Hitoshi KIHARA, Mitsuya NEzu, Tadao C. KATAYAMA,
Seiji MATSUMURA and Tomoo MABUCHI)

Genome analysis of Oryza species was continued this year. 25 interspecific hybrids were newly produced, among which 8 hybrids were cytologically investigated. The results of the investigation are summarizedin Table 1.
1)
2)This work was supported by Grant from Tokyo Seikagaku Kenkyiikai.
This work was supported by Grant RF 57080 from The Rockefeller Foundation.

RESEARCHES CARRIED OUT IN 1960

41

Table 1. Chromosome pairing in 8 interspecific Oryza hybrids
F I hybrids (¥xetl)
O. sativa indicaxO. stapfii andreciprocals

O. glaberrimaxO. perennis
O. australiensisxO. officinal isO. breviligulata X O. eichingeri
O. alta x O. officinalis
O. stapfiixO. latijolia
O. latijoliaxO. alta
O. malabarensis x O. latijolia

No. of cells
analyzed
90

50
100

14
12
13
12
14

Mode
12n
12n
24r

12
12
0~3

4n+ 28r

0~912n+ 12r

12~14

36r

o

24n
12n+24r

24
7~12

According to the table the genome constitutions of o. stapfii and O. alta
are AA and CCDD, respectively. The genome formulae given by MORrNAGA and KURrYAMA (1960) to O. sativa, O. perennis, O. glaberrima, O.
breviligulata, O. ojficinalis, O. latifolia and O. eichingeri have been
confirmed by our results. The genome of O. australiensis can not beeither C or A, because F I hybrids between this species and o. sativa had
24 univalents in the majority of PMC's as previously reported. Partial
homology between A and B genomes, as reported by KIHARA and NEZU
(1959), has been confirmed by the cross, O. breviligulata xO. eichingeri.
Concerning the genome constitution of O. malabarensis, the following
possibilities are considered; (1) one of malabarensis' genomes is homologous
to either C or D genome, or (2) each of its two genomes is partiallyhomologous to C and D. The first possibility is most likely, because 12
bivalents have been observed in the majority of PMC's in the F I hybrids
between this species and O. latifolia.

33.

Floating habit of 10 strains of wild and cultivated rice l )(By Hitoshi KIHARA, Tadao C. KATAYAMA and Koichiro TSuNEwAKr)
Resistance to flood is an important economic character of rice in tropical and subtropical countries. One of the factors determining it is thefloating habit. Using the floating rice samples collected by the membersof the National Institute of Genetics, the authors have carried out thefollowing experiment in order to elucidate the nature of this character.
10 strains belonging to Oryza sativa, O. perennis or O. glaberrima were1)This work was supported by Grant RF 57080 from The Rockefeller Foundation.

42

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

examined for their floating habit by sinking them up to 3m deep at 6
different rates, namely, 0 (control), 1.5, 3.1, 4.6, 6.2 and 9.3 cm per day.
Number of days of floating, plant height, leaf length, total internode
length, number of internodes and average internode length were observed.
The following results were obtained.
(1) The 10 strains employed can be classified into 3 groups regarding
floating habit. Strains 1 and 2 of O. sativa and strain 5 of O. perennis
are pronouncedly floating, strains 6 and 7 of O. perennis and strain 8 of
O. glaberrima are slightly floating, and strains 3 and 4 of O. sativa andstrains 9 and 10 of O. glaberrima are non-floating.
(2) In the pronouncedly floating rice an increase of plant height underinundated condition is almost entirely dependent upon the increase of thetotal internode length and very little upon leaf length. Elongation of
the latter is, however, rather important in the slightly floating rice.
(3) The increase of total internode length is almost equally dependent
upon the increase of internode number and the elongation of internodes.
(4) Rate of internode elongation increases, within a limit, proportionallyto the sinking rate. The floating habit of individual strains can becharacterized by 3 factors, a, band c which determine a regression of
the rate of internode elongation on the sinking rate. Factor a corresponds
to a point where the regression line crosses the vertical axis and indicatesthe rate of internode elongation achieved under normal conditions. It
can be used as an index of resistance against a sudden flood. Factor b
is a slope of the regression line and indicates the capacity of a strain toelongate the internodes in correspondence to rising water. The third
factor c is the maximum rate of elongation.
(5) Analysis of those 3 factors is a useful means for characterizing thefloating habit of individual strains. Among the 3 pronouncedly floatingstrains, strain 1 has an extremely large a and the smallest b, being mostresistant against a sudden flood. Strain 2 is characterized by the smallest a and c. Strain 5 has the largest band c, being most resistant forchronic inundation. All 3 slightly floating strains have only small a andc but their b's are comparable to those of the pronouncedly floatingstrains.

34. Production a/polYPloid wheat by nitrous oxide(By Hitoshi KIHARA and Koichiro TSUNEWAKI)

In 1954. Ostergren reported that N 20 treatment produced polyploids in
Crepis capillaris. The same treatment was applied by a few other Swedish
workers. The present authors tested Ostergren's method in a tetraploid

43

RESEARCHES CARRIED OUT IN 1960

wheat species, Triticum dicoccum Khapli (2n=28).
20 florets per spike were emasculated before flowering and artificiallypollinated two days after the emasculation. The spike bearing culms
were cut off at the third internode counting from the top 24 hours after
the pollination and treated with N20 gas for 5, 10 or 15 hours, each treatment at pressures of 3 and 6 atmospheres. The treated spikes were kept
in flasks containing a culture solution prepared after White's formula towhich glucose was added as a carbohydrate source in a concentration of
one per cent.
The seeds obtained from the treated spikes were placed on moist filter
paper in petri-dishes and allowed to germinate. Roots were collectedfrom each germinating seed, pretreated at O°C for 24 hours and fixedwith 3: 1 acetic alcohol. The root tips were stained with Feulgen's leucobasic fuchsin after hydrolysis with N-HCl for six minutes.
Effects of N 2G treatment on seed-setting, germination and chromosome
number are summarized in Table 1.
Table 1.

Effects of N 20 treatment on seed-setting, germination andchromosome number of Triticum dicoccum KhapliI[Success , No. of !
Proportion of(a); (b) , ofI plants:
I Seeds
Germi- Ipollina- IcytologUaneuset (%) I nation ltion (%)'cally ex_! diploid tetraploid
ploid
i
!
I(a)x(b)! amined 'I

Treatments--1~2-:-98.9

Control

3 atm's for 5 hrs.
II
II 10
II
II
II 15
II
6 atm's for 5 hrs.
II
II

10
II 15
II

II
II

I 76'5
I 74 . 4 I!
71.9 : 66.1
I 63.1 ! 83.2
80.7 I 70.8
78.6 I 86.3
69.3
72.2

:-78.3I

I

56 . 9
47.5
52.5
57. 1
67.8
50.0

I

60
56
59
55
50
62
47

100.0
100.0
30.5
16.4
100.0
19.4
2.1

0.0
0.0
45.8
72.7
0.0
37.1
46.8

0.0
0.0
23.7
10.9

0.0
43.5
51.1

Treatments for 5 hours did not induce any aberrant chromosome numbers. Treatment at both 3 and 6 atm. pressures for 10 or 15 hours produced tetraploids and aneuploids in 70 per cent of plants or more, obtained from treated flowers. The best success was obtained from the treatment at 6 atm. pressure for 15 hours, by which 98 per cent of treatedflorets gave polyploids and aneuploids.

44

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

35. PI monosomic analysis of Triticum macha(By Koichiro TSUNEW AKI and Hitoshi KIHARA)

Gene analysis of Triticum macha is important, because this species seems
to be the most probable progenitor of hexaploid wheats. It is also known
to have a normal allele of the third necrosis gene that has not yet been
identified.
The authors studied this species by crossing it to 21 monosomic lines ofChinese Spring. F 1 plants were grown in the greenhouse and theirchromosome numbers were determined from root-tip mitosis.
Due to semi-lethality of the hybrids, only a few F 2 seeds were produced
which did not allow to carry out the monosomic analysis to the F 2 generation. The results obtained in the F 1 generation are briefly reported.
Necrosis: All the F 1 disomics and 20 monosomics were necrotic withthe exception of mono-XVls, which were all normal. Among the 20
necrotic monosomics, the F 1 mono-Ills performed better as to plant height
and, especially seed setting than any other necrotic di- and monosomics.
These findings indicate that chromosomes XVI and III of Chinese Spring
carry the third complementary necrosis gene and a modifier that conditions severe necrosis, respectively.
Growth habit: Monosomic F 1 plants for either chromosome IX or XVIIIof Chinese Spring had winter growth habit, indicating that those chromosomes of T. macha carry recessive genes controlling this habit. They
seem to be the same as those found in other hexaploid wheats.
Awnedness: Monosomic F 1 plants for chromosome VIII or X wereawnletted, whereas all the other hybrids were awnless. This result indicates that T. macha carries hd and b2 on chromosomes VIII and X,
respectively.
In comparison with Chinese Spring, T. macha seems to have the following genes for the three characters studied so far:
Character (Responsible chromosome)
Necrosis: -Growth habit! - A;;edness
I

V

Chinese Spring

T. macha

I

XIII

XVI

I

XVIII

-\--::-1- :-1 ~:: ~::c i-- -- - - - - - - - - - - - - - - - - - - - - - - - - - -

IX

VIII
Hd
hd

X

RESEARCHES CARRIED OUT IN 1960

36.

45

Munosomic and conventiunal analyses of glume hairinessand ear density in 8 varieties of common wheat(By Koichiro TsuNEwAKI)

Inheritance of glume hairiness and ear density of 8 agronomic varieties'of common wheat, Triticum aestivum was studied by monosomic andconventional analysis. Among them, Elgin and Red Egyptian belong tosubspecies compactum and Chinese Spring, Prelude, Red Bobs, S-615, Jones
Fife and Kharkov to vulgare. Concerning the hairiness of glumes,
Prelude and Jones Fife are pubescent and the other 6 varieties are nonpubescent.
The results obtained by the two methods were in complete agreementwith each other. They are summarized as follows:(1) Glume hairiness is controlled by a single dominant gene, Hg,located on chromosome XIV. Genotypes of Jones Fife and Prelude andthe other 6 varieties are Hg Hg and hg hg, respectively.
(2) Spike density characterizing the subspecies compactum is determinedby spike length but not by the number of spikelets per ear. Short ear
length of compactum is controlled by a single dominant gene, C, locatedon chromosome XX. Genotypes of Elgin and Red Egyptian and the othersix varieties are CC and cc, respectively. The different spike lengths
found among vulgare varieties are associated with the number of spikelets
per ear and are controlled by polygenes.

37.

Monosomic analysis of synthesized hexaploid wheats(By Koichiro TsuNEwAKI)

Polyploidy is a prevailing phenomenon in higher plants. For a study ofthe evolutionary significance of polyploidy, a comparative gene analysisof amphidiploids and their ancestral species is a useful approach. Furthermore, this type of work will bear informations on the origin of genes now
present in the amphidiploids.
A comparative gene analysis of common wheats and their ancestors,
Emmer wheats and Aegilops squarrosa, is now possible, because (1) anumber of hexaploid wheats have been synthesized from various speciesof Emmer wheats and various types of Ae. squarrosa and (2) the monosomic series of a common wheat established by Sears has made gene analysis of hexaploid wheats very easy.
A large amount of informations have been accumulated on the chromosome locations of genes which control morphological and physiological

46

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

characters of common wheats. In order to get comparable informations
for Emmer wheats and Ae. squarrosa, the author crossed 21 monosomic
lines of Chinese Spring (common wheat) to 5 synthetic hexaploid wheats
listed in the following:
Synthetic components
Synthetic 6x

SourceEmmer wheat

Ae. squarrosa

Kihara's No.1 I T. dicoccoides

ABD-l

"

2

"

"

3

Sears'

No.3

spontaneo-nigrum
T. turgidum

Ae. squarrosa No.2

T. dicoccum Vernal
T. durum melanopus

Ae. squarrosa (Sears')

11

Golden Ball"
" 4
T. durum hordeijorme
5Carleton""- - - - - - - - - - _ -----_._-,
..

...

I

"
"

, - - - -The results obtained from the Fl generation of those crosses suggest
the following possibilities for the origin of genes controlling growth habit
ahd awnedness of common wheats.
Growth habit. Sgl and sgz genes controlling winter growth habit ofcommon wheats have been derived, respectively, from winter Ae. squarrvsa
and winter Emmer species such as T. dicoccoides. 5g2 gene known to bepresent in early spring varieties of common wheats has been originatedfrom early species of Emmer wheats, such as T. turgidum and T. dicoccum. 5g2 C gene present in late spring varieties of common wheats has
been originated from late spring varieties of Emmer such as T. durum
varieties. 5g2 and 5g2c genes of Emmer wheats appeared to be epistatic
to Sgl gene of Ae. squarrosa.
Awnedness. 2 awn-promoting genes, al and az of common wheats, havebeen originated from Emmer wheats and Ae. squarrosa, respectively. 3
inhibitors, Hd, B 2 and A 4 present in Chinese Spring are likely to havearisen at the hexaploid level.

38.

Marker lines in Triticum aestivum var. 5-615(By Koichiro TSUNEW AKI)

In order to establish isogenic marker lines of individual chromosomes
in common wheat, the author is carrying out a project to isolate major
genes from known sources and to bring them into the same genetic
background by successive backcrosses. An awned, spring vulgare variety,
S-615, has been chosen as the recipient variety. The present stage of47

RESEARCHES CARRIED OUT IN 1960

project is represented in the following table:
In order to mark the other 9 chromosomes, on which no major genes
have been found, artificial production to trans10cations between Hp geneof rye and wheat chromosomes has been attempted by irradiating a wheatrye addition line provided by Dr. L. E. Evans.
Chromosomes]
Symbols
marked I- - - --------

III
IV
V
VIII
IX
X
XI
XIII
XIV
XVI
XVIII
XX
11
21

39.

Characters
controUed

Donorvarieties,

I

No. ofbackcrossesI
made-----~~·~--~I~~-

v
Hp

Ne!
Hd
B!
Bz
He
Nez
Hy
sp
sy!
C

I

virescenthairy peduncle
necrosishooded awn
awn suppression"

red coleoptile
necrosisihairy glume
sphaerococcum
characters
I winter growthicompact ear

Chinese Spring 11
Rye 21PreludeChinese Spring
Jones Fife
Chinese Spring
Hope 11
Kharkov
Jones Fife
T. sphaerococcum
Kharkov
Elgin

I

I

1
1
1
2

2
2
1
2
2
2
2
2

Obtained from Dr. E. R. Sears
Originated from CorneU University

An early ecotype of Agropyron tsul?'ushiense var. transiens Ohwi
(By Sadao SAKAMOTO)

A. tsukushiense var. transiens, that grows in fields or along road-sides,
1S very common and widely distributed in Honshu, Kyushu and Shikoku.
It is also known in China, Manchuria and Korea. This species is hexaploid (2n=42) and shows a wide variation in many characters. A strain
which differs in several characteristics from the common strain (designatedas common type) was found in the vicinity of Misima. After ecologicaland genetical studies of this strain, it was recognized as an ecotype ofA. tsukushiense var. transiens.
Characteristics of this ecotype which differ from those of the common
type are as follows: 1) This ecotype was found as a swarm in the idle
lying paddy fields in the valleys of hilly regions. 2) It is strongly waxy
and deeply anthocyan-pigmented. 3) The upper side of rosette lewes ispubes::ent. 4) Plant height, the first internode from the top, fhg leaf

48

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

and spike are shorter. Number of spikelets is much smaller. 5) Empty
glume, lemma and palea are longer, but awn is shorter. 6) Seeds arelarger and heavier. 7) This ecotype first flowers at the end of April,about 25 days earlier than the common type.
Hybrids between the ecotype and the common type were easily obtainedby artificial pollination and their growth was very vigorous. Out of the
19 characters of the FI'S, 14 were intermediate between these of theparents. At meiosis of F I hybrids, a quadrivalent was observed in 14 %of 163 metaphase plates. Pollen-fertility and seed-fertility of the FI's were
77 % and 62 %, respectively, being lower than those of both parents.
The germination and growth of the F 2'S were very good, and all plants
were fertile. Eight characters, namely, pubescence of rosette leaves, colorof anthers, date of flowering, plant height, length of the first internode,flag leaf and spike, and number of spikelets, were observed in the P, F I, F 2
and the first backcross generations. The mode of inheritance of the firsttwo characters showed a 3: 1 segregation in the F 2's, and no linkage wasfound between them. Segregation of the other six characters was typically continuous, suggesting that those characters were controlled by alarger number of genes. Among these six characters, genetic correlations
were observed in the F2'S. Negative correlations were found between thedate of flowering and the other five quantitative characters, while thecorresponding correlations were positive in the parents.
Among several characteristics which distingush the ecotype from thecommon type, the following three are the most important:
1) Differentiation in the growing habitat.
2) Genetic differentiation relating to some quantitative characters.
3) Difference in the flowering time.
Since the earliness of flowering was strikingly characteristic, this ecotype
was designated as "early ecotype".
The early ecotype was often found together with Chinese milk-vetch(Astragalus sinicus L.) in paddy fields. Its distribution seems to followthat of Chinese milk-vetch.

40.

Photoperiodic responses of Oryza species IIft)(By Tadao KATAYAMA)The present author had already reported his investigation in rice on
the influence of plant age on photoperiodic sensitivity. In this year, an
experiment was carried out in order to study the following three points;(1) relationship between photoperiodic sensitivities of various Oryza species~_._-----------

1l

This work was supported by Grant RF 57080 from The Rockefeller Foundation.

RESEARCHES CARRIED OUT IN 196049

and latitude of their habitats, (2) photoperiodic sensitivities of various
species which occur in the state of nature in the same latitude, and (3)effect of twilight on photoperiodic responses. The results of the experiments are briefly presented.
(1) Among photoperiodically sensitive strains belonging to various Oryza
species, whose habitats vary between lOON and 35°N, a high negative
correlation was found between the latitude of the habitats (X) and heading dates expressed by number of days from seeding to heading (Y),when they are grown at Mishima (35°N). The relationship between X
and Y can be expressed as follows:
Y=185-3.0X(2) The cultivated ones become sensitive to short-day treatment at an
earlier plant age than the wild ones in the following order:

O. sativa<O. glaberrima~O. sativa spontanea<O. perennis(3) Near the critical day length, twilight given after dark periods
delays heading in all three species, O. sativa, O. glaberrima and O. perennis.
On the other hand, the same twilight given after light periods delays
heading of some species but does not affect that of others.
41.

Studies on the intercellular spaces in rice
(By Tadao KATAYAMA)On the aerating system of roots of Oryza sativa and its physiologicalsignificance, several reports have been published.
As a quantitative determination of the total intercellular air space inrice was lacking, the author investigated the variations of its volume inrelation to cultivation procedures and developmental stages.
Two kinds of Japanese rice, a paddy rice and an upland variety were
l1sed as materials. Two other crop plants, Zea mays L. and Vicia faba
L., were also used for comparison.
Computations were made by the following formulae:
GI __ G2V =-s

I
f th emaenaSlnmm
t' I'
3voumeo

G G=--s-·. t erce11 u I ar space III. mm 3
voI ume 0 f th e III

v% =-~ X 100

volume percentage of the intercellular space,

V

3-

2

G 1 •••• weightin air, G2 ••• • weight when submerged in water, G3 ••• • weight when infiltrated and submerged in water S . ... apparent specific weight of water used (=[specific weight of water]-[specific weight of air])

50

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

1) The intercellular spaces of root are larger in rice than in corn andbroad bean. The paddy varieties develop larger air canals than the uplandones when raised under the same conditions. The root tissues attain theultimate volume at some distance from the root tip varying in accordance
to the variety and culture conditions.
2) The development of air canals in the roots of rice is markedly influenced by cultivation conditions especially with respect to the air content
of the soil. The volume of the total air space is three times as large in
non-aerated as in aerated soil.
3) The structure of the leaf and the volume of its total air space varywith the ascending position of the leaf. Differences were also observed invarious parts of a leaf.
4) The intercellular spaces are larger in the blade than in the sheath ofyoung leaves but such relationship cannot be clearly seen in older leaves.
5) The total intercellular space of the leaf is larger in paddy rice thanin upland rice at young seedling stage but the relation is not clear atlater stages.
6) The volume per cent of air space in the leaf is also influenced byenvironmental conditions of the soil. In the paddy it is larger than in
the upland field. The differences are especially clear in young seedlings
and become later obscure.
7) Such remarkable adaptability of rice to moisture and aeration may
explain why this crop can be grown either in paddy or upland fields.

42.

On the mechanism of the appearance of gigas-plantsfrom mullisomic dwarf wheat(By Tomoko OHTA and Seiji MATSUMURA)

In the offspring of the pentaploid wheat hybrid between Triticum Spelta
and T. polonicum dwarf plants possessing 40 chromosomes (20n) arefound. They are nullisomics, deficient in a chromosome pair or the
D-genome. As it is the a~g-chromosome in the D-genome which is missing, they are called a~g-dwarfs. So-called giant plants of normal heightand vigor appear unexpectedly in their selfed progeny. These are respectively called a~g-gigas, according to the original seven different dwarfs,
in which the missing D-pair is replaced by the homoeologous A- or B·
chromosome pair.
Gigas-plants are assumed to appear from nullisomic dwarf wheat as aresult of chromosome aberrations in MI. Various chromosome aberrations
in MI and unequal distribution in AI of a~g-dwarf lines were observedand chromosome aberration rate, theoretical and observed appearance
rates of gigas were compared for seven dwarf lines. Except the g-dwarf

RESEARCHES CARRIED OUT IN 1960

51

(Dwl. 5), theoretical appearance rates of gigas are too small against theobservation, amounting to 5--10 %. There are many factors involved,
such as competitive fertilization, elimination of dwarfs and others. Still
more important is the fact that the special chromosomes which arehomoeologous to the deficient one, become easily aberrant. In the caseof b-dwarf, the special chromosome is a Sat-chromosome which becomesvery often a univalent.

43. Cytological and histological observations onmale sterility in sugar beets(By Tomoko OHTA and Seiji MATSUMURA)

Two cases of male sterility in sugar beets (Beta vulgaris) were cytologically and histologically investigated. One case represents physiologicalmale sterility caused by low temperature. In the investigation of thiscase, each beet of variety GW 359 was longitudinally cut in two pieces.
One half was subjected to low temperature treatment (5°C) for 24 hours,
two weeks after the first flowering. The other half was untreated andused as control. Young anthers had a normal appearance before meiosis,
but some effect was observed during this stage and giant pollen grains
were found. On the other hand, various abnormalities were found in theanthers treated in the stage between pollen tetrad formation and maturepollen grain production, especially when the pollen had developed rapidly
absorbing nutrients from the tapetal tissue. The pollen sterility increased
markedly.
The other case represents genetical male sterility. One completelymale-sterile plant was found in a commercial hybrid variety, US 216 malesterile x US 226. In its anthers the tapetal cells developed abnormallyand remained longer intact than in the normals. The pollen grains, notbeing able to absorb nutrients from the tapetum, finally degenerated. In
this case male sterility seems to be cytoplasmic.
In both cases abnormal development of tapetal cells was most striking.
The relation between pollen grains and the tapetal tissue is thought
to be the most important cause of male sterility (d. Seiken ZiM No. 11).

44.

Variation in susceptibility to blast fungus
in cultivated and wild rice l )(By Keizo KATSUYA)

13 strains of wild rice, Oryza sativa f. spontanea, O. perennis and O.
11This work was supported by Grant RF 57080 from the Rockefeller Foundation

52

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

breviligulata, and 16 strains of cultivated rice, O. sativa and O. glaberrima,
were tested, concerning variability in susceptibility to blast fungus,

Piricularia oryzae.
Localities where the seed samples were collected were:Table l.

Africa, Ceylon,

Average value and genetic variability in susceptibility to
blast disease in strains of wild and cultivated rice... _ - - - - - _ .. -

- ~ _

-----

Species

Locality

Averagesuscepti bility*

Genetic
variability ('Yo)

Oryza glaberrima
O. glaberrima

Africa

0.708
0.936

45.28
44.04

O. sativa
O. sativa
O. sativa
O. sativa
O. sativa
O. sativa
O. sativa
O. sativa
O. sativa
O. sativa
O. sativa
O. sativa
O. sativa
O. sativa

Burma

0.598
0.848
0.608
0.698
0.592

10.53
85.36
17.24
17.56
59.32

-----_... _..

_-

"

"

India

"

Malaya"Thailand"Taiwan

"

Japan

"

Ceylon"O. breviligulata
O. breviligulata

AfricaO. sativa f. spontanea
O. sativa f. spontanea
O. sativa f. spontanea
O. sativa f. spontanea
O. sativa f. spontanea
O. sativa f. spontanea
O. sativa f. spontanea
O. sativa f. spontanea
O. perennis
O. perennis

0.576
0.604
0.578
0.694
0.730
0.650
0.616
1.446
1.507

0.00
10.45
38.65
85.60
17.94
9.57

0.845
1.087

95.54

Ceylon

4.498

India

0.594
7.178
0.578
0.823
0.642
0.622

"

1.491

92.27
17.81
22.62
25.42
87.09
32.43
38 . 76
99.79

India
Thailand

0.648
0.746

98.93
32.43

"
"

Malaya"ThailandTaiwan----- - - - - _ .. _- --* The estimate for the most resistant plant being 0.5.

1

RESEARCHES CARRIED OUT IN 1960

53

India, Burma, Malaya, Thailand, Taiwan and Japan. The plants weregrown in pots. Susceptibility to the disease was tested by the leaf-sheath
method using the leaf sheath of the second youngest leaf. Testing wasdone twice for each individual plant.
The P-2 strain of blast fungus
was used. A verage susceptibility degree and genetic variability for eachstrain are shown in Table l.
Some strains of O. sativa f. spontanea collected in India and Ceylon
were highly susceptible. o. breviligulata collected in Africa, O. sativa f.
spontanea in Taiwan and O. sativa in Ceylon were considerably susceptible. Other strains were resistant. It is suggested that in general thestrains distributed in Ceylon are susceptible, while those in Malaya andThailand are resistant.
Table 1 shows that most strains of wild rice and a few strains of cultivated rice were highly heterozygous with respect to blast susceptibility.
Those occurring in Africa seem to be considerably heterozygous, while
strains from Thailand and Japan were much less heterozygous.

45.

Variability of seed size in wild rice populations of
Burma and some African countries(By Kan-Ichi SAKAI, Shin-ya IYAMA and Takashi NARISE)

Variability of seed size was measured for several populations of wild
rice collected in Burma and a few African countries. The number of
populations collected was five for Oryza perennis, seven for O. sativa f.
spontanea, six for O. breviligulata and four O. barthii. The former two
species were from Burma and the latter two from Africa. A number ofpanicles were collected at random from each population, and ten seeds
from each panicle were measured for their length and width. The analysis
of variance of data was conducted for each population separately. Estimated values of mean length and width, environmental and genotypic variances, the proportion of genotypic variance to phenotypic variance, andgenetic and environmental correlations between seed length and width aswell were compared among different species and different strains.
Though a detailed discussion and concluding remarks will have naturally
to await the completion of this serial work, a few facts of interest may
be pointed out. The first thing to be mentioned is that the wild ricespecies, O. breviligulata and O. barthii, growing in Africa have on anaverage bigger seeds than O. perennis or O. sativa f. spontanea found in
Asia. Secondly, wild population in Africa are genetically highly heterogeneous with regard to seed size, the heterogeneity being comparable tothat of wild rice strains found in Asia. In this respect, it is of interest to

54

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

find that wild populations of O. perennis growing in Burma are often
unusually homogeneous. Genetic correlation between seed length and seedwidth is very variable, positive in some strains and negative in others,
while environmental correlation between them is very low throughout.

46.

Comparative studies on growth rate of seedlingsin wild and cultivated rice
(By Kan-Ichi SAKAI and Shin-ya IYAMA)

Growth rate of seedlings was compared between cultivated and wild
strains of rice. The number of varieties of cultivated rice used in thisexperiment was ten for Oryza sativa, nine for O. glaberrima and thenumber of wild strains was thirteen involving O. sativa f. spontanea, O.
perennis and intermediate types between them. Growth rate was measured by seedling length between the second and the fourteenth day after
germination.
It was found from this experiment that cultivated as well as wild strains
were highly variable in respect of growth rate of young seedlings. The
cultivated varieties however had in general a higher growth rate in theseedling stage than the wild strains. Experiments are now under progressfor further study.

47.

Seed growth in cultivated and wild rice
(By Takashi NARISE and Kan-Ichi SAKAI)

The growth of seeds of cultivated and wild rices was investigated.
Eight strains of cultivated rice including Oryza sativa and O. glaberrima
and twelve of wild rice including O. perennis, O. sativa f. spontanea andO. breviligulata were used in this study. Observation was made in thefield and in the greenhouse. One to 29 days old spikelets after anthesis
were collected from each strain, and their fresh weight, dry weight andwater content per seed were measured.
In cultivated rice, the total fresh weight of seeds generally increased
up to the 24th day after fertilization in O. sativa and 22nd day in O.
glaberrima, whlie dry weight increased up to the 22nd day fertilizationin O. sativa and 20th or 22nd day in O. glaberrma, the water content increasing up to the 13th day in both species. In wild rice, an increase in
the total fresh weight and dry weight was observed up to the 10th day
in O. sativa f. spontanea and to about the 11th day in total fresh weightand 14 day in dry weight in O. breviligulata. The analysis of varianceindicated that there was no significant difference in fresh weight between

RESEARCHES CARRIED OUT IN 1960

55

cultivated rice species, but a significant difference in the deY weight, thesativa strains having heavier seeds than the glaberrima strains.
The growth rate of seeds measured in terms of daily increase in freshas well as dry weight was compared between wild and cultivated speciesof rice. The same comparison was also made among strains of either
cultivated or wild rices. It was found from this comparison that thegrowth rate of seed per day was not different among various species,
whether wild or cultivated. It is concluded accordingly that the only
defference between wild and cultivated rice species in respect of seed
growth may be that wild species complete ripening several days earlier
than cultivated species, producing smaller kernels than the latter: 23
days were required for O. sativa to ripen, 21 or 22 days for O. glaberrima
and only 10 days for wild rices.

48.

Survey of variation between Oryza breviligulata andO. glaberrima collected from West Africa(By Hiroko MORISHIMA and Hiko-Ichi OKA)

An African wild rice species, O. breviligulata, may be regarded as theprogenitor of O. glaberrima which is cultivated in West Africa, since thetwo species have many common characteristics and fertile F 1 hybrids.
With the view to investigating the variation between wild and cultivatedforms in these two species, a number of strains belonging to them, collected by Dr. K. FURUSATO of this Institute from West African countries,
were grown in a short-day field in Mishima, and data were taken forvarious characters. By choosing typically wild (breviligulata) and cultivated (glaberrima) strains, first a discriminant function for classifying wild
and cultivated forms was constructed. Measurements of degree of grain
shedding, length/width ratio of spikelet, spikelet number per panicle andrachis number per panicle were combined to make up the discriminant
formula. Regarding the scores given by this formula, the strains tested(49 in total number) showed a continuous series of intergrades between
typically wild and cultivated forms. This pattern of variation from wild
to cultivated forms appeared to be similar to that previously observed by thewriters in Asian materials including O. perennis and O. sativa (the so-called
O. sativa f. spontanea is included by us in O. perennis as one of its forms).
Secondly, variations of six characters among 37 strains of O. breviligulata
were studied by factor analysis of correlations. The first factor extracted
from the multivariate variation, with a 39 % contribution, was found toindicate the variation in the direction of cultivated type. The second
factor made a very small contribution (3.6 %) and did not show a definite

56

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

biological significance. This result markedly differs from the variation in
Asian populations of O. perennis, in which the first factor showed thedifferentiation into perennis and spontanea types and the second factor
showed the variation in the direction of cultivated type (reported in Annual
Report No. 10: 49-51, 1960).
Thirdly, in order to examine how the variation in the direction of cultivated type is related to each character, variation patterns in wild (breviligulata) and cultivated (glaberrima) forms were compared with each otherregarding various characters. Significant differences between the twoforms were found in spikelet width, length/width ratio of spikelet, awn
length, degree of grain shedding and apiculus hair lenght. Except for
the last one, these are the characters differentiating wild from cultivatedforms in the group of O. perennis and O. sativa. In spikelet length,
length/width ratio of spikelet, awn length and ligule length, the wildforms (breviligulata) had wider range than the cultivated forms (glaberrima).
In contrast, in apiculus hair length, rachis number per panicle and spikelet
number per panicle, cultivated forms a wider range. This pattern ofvariation does not consist with what is found between O. perennis andO. sativa.
49.

Parallel variation from wild to cultivated forms
in Sativa and Glaberrima Series of Oryza(By Hiroko MORISHIMA and Hiko-Ichi OKA)

The wild progenitors of O. sativa and O. glaberrima are thought to be
O. perennis (including O. sativa f. spontanea assumed to be a form of O.
perennis) and O. breviligulata, respectively. The Sativa series (including
O. sativa and O. perennis) and the Glaberrima series (0. glaberrima andO. breviligulata) can be discriminate~ by ligule shape and other characters,
and their F 1 hybrids are highly sterile. This study was an attempt to find
how the variation from wild to cultivated forms which might have occurredin parallel in the two series of species can be analysed statistically, sinceparallel variation is regarded to be an obstacle in numerical systematics.
Correlation coefficients between strains were calculated from measurementsof 20 quantitative characters taken for 20 strains belonging to the above·
mentioned four species (five strains for each species). In addition to this,discriminant scores were computed for the 20 strains from a discriminant
formula for classifying the Sativa and Glaberrima series, and a matrixof the discriminant scores was subtracted from the matrix of above-mentioned correlation coefficients. In this process, the discriminant scores
were adjusted so that 25% of the total multivariate variation was sub-

RESEARCHES CARRIED OUT IN 1960

57

tracted. Then the residual matrix was analysed by factor analysis (averoid) to find unknown variation factors. The first factor thus extracted
from the residual matrix was found to indicate the differentiation of strains
into wild and cultivated forms, spending 38% of the total variation. By
scattering the strains tested in the plane defined by the discriminantfunction and the first factor axis, the parallel variations from wild tocultivated types in the Sativa and Glaberrima groups were compared.
Among the 20 strains tested, the variation in the Glaberrima series appeared to be continuous, but in the Sativa series the wild and cultivatedstrains could be separated.

50.

Further studies on hybrid sterility relations among cultivatedand wild strains of rice
(By Kokichi HINATA and Hiko-Ichi aKA)

We have reported last year (Annual Report No. 10: 51-53, 1960) thatmost Asian wild rice strains of Oryza perennis and O. sativa f. spontanea
show a high fertility in F 1 hybrids with different test-strains belonging to
Indica or Japonica type of O. sativa, and that a clear differentiation into
the two types was not found in wild plants. This investigation was continued and more materials were observed. The results were examined byscattering the newly tested strains on a diagram introduced in our lastreport, in which the axes of abscissa and ordinate represented the firstand second component axes extracted from the data for cultivated rice
varieties. The diagram after addition of 11 new strains still showed thesame features of variation as pointed out last year.
Further, a number of strains belonging to O. glaberrima and O. breviligulata, obtained from West Africa, were crossed with a set of test-strains(consisting of O. sativa and O. glaberrima) to examine their hybrid sterility relations. The majority of crosses between glaberrima and sativa,
more than 200 in total number, did not show a pollen fertility higher than
3%. However, in crosses with a few sativa strains from India and Viet
Nam, it amounted to 5 % to 8 %. The wild strains of sativa type (0.
perennis and O. sativa f. spontanea) showed relatively high pollen fertilities in hybrids with O. glaberrima, the highest reaching 25 %.

51.

Hybrid swarms between wild and cultivated rice species,
Oryza perennis and O. sativa(By Hiko-Ichi aKA and Wen-Tsai CHANG)

Several wild rice populations supposedly of hybrid origin between wild
and cultivated forms were analysed. First, Patna population, found in the

58

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

suburb of Cuttack, Orissa, India, though it appeared to be a mixture ofwild and cultivated plants growing in a paddy field, was shown to be a
hybrid swarm. A part of its plants, having many wild characters and a
few cultivated ones, were found to be highly heterozygous for genes ofcultivated rice. Secondly, Sampatoon population, found in a stream running through paddy farms of glutinous rice in the suburb of Chiengmai,
Thailand, was observed. It consisted of plants of intermediate perennisspontanea type, and contained many heterozygotes for the glutinous gene.
The offspring of the heterozygotes tended to be in various charactersintermediate between the wild and the cultivated type. It was also foundthat, though in northern and north-eastern parts of Thailand, glutinous
rice is grown in almost all paddy farms, the majority of wild populations
growing in proximity of glutinous rice fields had a low frequency of theglutinous gene, so that a hybrid swarm might be regarded to be rather
rare. Thirdly, three small populations found in Tao-Yuan Prefecture,
Formosa, which were phenotypically of perennis type, were observed.
They were found to be highly heterozygous for genes of cultivated rice.
Thus, it was pointed out in general that a hybrid swarm could preservea great amount of genetic variability, which might cover the whole rangeof variation from the wild to the cultivated type. Based on the resultsof these observations, emphasis was laid on the significance of hybridization in the process of creation of cultivated rice. (Published in Evolution, in press)

52.

A Survey of drought resistance in wild and cultivatedrice strains(By Hiroko MORISHIMA and Hiko-Ichi OKA)

In Oryza sativa, the so-called Indica varieties are generally more resistant to drought than the Japonica varieties (OKA 1953, 1958). An experiment was made to compare this character between several wild and cultivated Oryza species. A number of strains belonging to O. sativa (21strains), O. perennis (42 strains, including O. barthii and O. sativa f.
spontanea), O. glaberrima (29 strains) and O. breviligulata (29 strains) were
tested by the Mimosa method. By this method, the drought condition ina pot is judged by the suspension of stimulation movement of a Mimosa
pudica plant grown together with the plants to be tested. Thus the resultshows the ability of plants to survive a strong drought. In O. sativa, as
was previously known, strains of the Indica type were found to be generally more resistant than those of the Japonica type. Strains belonging
to O. perennis showed a wide variation ranging from highly to very weakly

RESEARCHES CARRIED OUT IN 1960

59

resistant, while those of the spontanea type were generally resistant.
Strains of O. glaberrima and O. breviligulata were generally weak andmost of them did not survive the test.

53.

Comparison of growth and fertilizer response between
Oryza sativa and O. glaberrima(By Kokichi HINATA, Hiroko MORISHIMA and Hiko-Ichi aKA)

Five sativa and five glaberrima varieties, early enough to be harvested
before October frost in MISIMA, were tested in a field experiment withthree fertilizer levels. A strain of O. sativa f. spontanea from India wasadded to this experiment, but it was too late to observe its yielding capacity. The experiment was designed as latin-square split plots with three
replications, in which sub-plot represented a variety. Fertilizer treatment
consisted of zero, standard (12 gm/m 2 ) and double nitrogen levels with thesame amount of phosphoric acid and potash (both being 8 gm/m 2 ). Seedlings raised in a nursery-bed were transplanted at normal (25 cm x 25 cm)
and high (12.5 cm x 12.5 cm) density.
The growth of plants in the normal-density plots was traced by the" growth analysis" method. It was found that the varieties of O. glaberrima generally had smaller "net-assimilation rate" than those of O.
sativa. However, they had wider and thinner leaves than O. sativa, or ahigher regression of leaf area on leaf weight. The large leaf area may
compensate for the low assimilation rate resulting in the" relative growthrate" of this species approximately the same as that of O. sativa. This
relation seemed to hold good at least in the earlier stages of growth when
the leaves are not crowded. When the "leaf-area index" becomes highin later stage, especially in densely planted plots or in highly fertilized
plots, O. glaberrima might have a lower assimilating power than O. sativa.
It may then have shown smaller plant weight and smaller panicle number
at maturity.
Regarding fertilizer responses, the Japonica varieties of O. sativa had
a higher rate than the Indicas as is well known. In O. glaberrima, fourof the five varieties tested showed a low rate as that of Indicas, but theremaining one had a high rate as that of Japonicas.
54.

Inheritance of mono and polyembryony in Citrus(By Kazuo FURUSATO)

Ten years ago, in the Experimental Citrus Farm in Kuzura, crosses
have been carried out between mono- and polyembryonic Citrus varieties.

60

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

They were as follows:
Monoembryonic
C. Tamurana
C. sulcata

Polyembryonic
C. natsudaidai
C. sinensis
C. Kinokuni
C. reticulata
C. leiocarpa
C. k6kitsu

The F , could be examined now, after ten years, and the results are
given in the following table.
Table 1.
Variety name

Cross
---_

..

mono.

Number of
F , plants

Polyembryonic

Monoem·bryonic

2

o

2

_----

C. Tamurana

X

X

mono.

C. sulcata---------- - --1--------------------\

c. TamuranaX

C. natsudaidai
C. Tamurana
mono.
X

poly.

X

16

13

3

2

2

o

1

1

o

2

1

C. sinensis
C. Tamurana
X

C. Kinokuni
C. sulcata-.-----+~: :;~~"'cii----l-X

poly.
X

Ie
' IataI· retwupoly.

C. k8kitsu
X

1

.

3

4

i

----1-o.
1

3

C. natsudaidai

The number of F , plants is of course small but it may be concluded
that the inheritance of this trait is controlled by an allele pair, mono·versus polyembryonic. The former allele is recessive, the latter is dominant. The two monoembryonic varieties must be recessive homozygotes.
Of the polyembryonic strains C. natsudaidai could be a dominant homozygote. As to the other polyembryonic varieties the small numbers do
not allow to draw a decisive conclusion as to their homo- or heterozygotic
nature.

RESEARCHES CARRIED OUT IN 1960

55.

61

Breeding of amphidiploids from a hybrid between
Nicotiana rustica and N. tabacum(By Kazuo FURUSATO)

A cross between Nicotiana rustica and N. tabacum is usually very difficult, and the seeds obtained from either direction of the cross do not
germinate.
In the spring of 1959 the cross with N. rustica as the female parent
was repeated in a greenhouse kept at low temperature. From 1500 flowers
pollinated 1300 capsules were obtained which yielded 134 viable seeds.
But from the reciprocal cross, N. tabacum x N. rustica, carried out under
the same conditions no viable seeds were obtained.
The F 1 seedlings were treated with colchicine and three tetraploid
plants were obtained.
Pollen fertility in F 1 was about 1%; it amounted to about 89% in thetetraploids. But neither tetra- nor diploids produced capsules by selfpollination. Only when the tetraploids were backcrossed to N. tabacum,
Table 1.
Leaf
Largest leaf
Average weight
'
I
I
Stem
, P ant I thO k.1 of raw leaves
I
I height
n~~s - number L~ngth width position I from one plant

SpeciesI

N. tabacum

(cm)1

108.8

(Bright Yel·low)
4x (N. rustica

I

(cm)1

2.2

(cm)

21.2

44.8

(g)
7.4

233

6.2

480

I

I

X N. tabacum) ,X
N. tabacum

(cm)

24.0

I

131.4

3.0

20.3

50.8

32.8

- - - - _I. _ - -Table 2.
SpeciesI

Rustin(:Yo)

Nicotine
(:Yo)

2.33
0.69

0.92
0.72

0.94
1.44

1.44

0.96

3.53

Chlorogenic acid i(:Yo)---,---,---,---- ,--------------

N. tabacum (Bright Yellow)
N. rustica (Brazilia)
4x (N. rusticaxN. tabacum)
X

Bright Yellow

III- - - - -In Table 1 a few characters are given for Bright Yellow and for the 4x hybrid
backcrossed to Bright Yellow.
In Table 2 the results of the chemical analysis are given for Bright Yellow,
Nicotiana rustica and 4x hybridxBright Yellow.

62

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

capsules were produced, and the seeds were viable.
tained on the average 80 seeds.

The capsules con-

56. Selection for low nicotine content(By Kazuo FURUSATO and Akira MIYAZAWA)

Synthetic tobacco obtained from a hybrid between Nicotiana sylvestrisand N. tomentosiformis was crossed with the commercial variety Bright
Yellow and selfing as well as backcrossing were practiced for five years.
Yearly selection for nornicotine finally yielded 29 plants with high nornicotine and very low nicotine content. In addition, 3 plants among themlacked the cherry red leaf character.
The results of the chemical analysis of several plants were as follows:
Table 1.
StrainNicotine (%)

Fe 33
Fe 35
Fe 2
Fe 99
F e 123

0.28
0.28
1.72
0.93
0.70
2.38

H-

57.

8

Nornicotine (%)

1.32
0.50

0.37
0.26

0.49
0.22

Heterotic vitality in natural populations of Trzllium
kamtschaticum Pall

(By Yuichiro HIRAIZUMI, Takashi NARISE and Ichiro FUKUDA)

Vitality, one of the major components of fitness, was studied in natural
populations of T. kamtschaticum. The main points studied were as follows.
1. The f value computed for chromosome E was chosen as an estimate
of inbreeding coefficient for each population. This was on the assumption that this chromosome was relatively neutral regarding vitality.
2. With this f value, a method was proposed to measure the vitalityof genotypes when only data from natural populations are available.
3. The method was applied to the actual data obtained from natural
populations of T. kamtschaticum and it was concluded that the heterotic
vitality is a general phenomenon in natural populations of this plant;
if two (possibly more) types showing especially higher vitality in theirhybrid are introduced or produced, they will be accumulated in populations to form the "specific local color."

RESEARCHES CARRIED OUT IN 1960

58.

63

Cytogenetic studies in the genus NICOTIANA XIII
(By YO TAKENAKA)

The reduction division in PMC's was studied in 3 interspecific hybrids:
N. suaveolens x N. longiflora, N. suaveolens x N. plumbaginifolia and 4 x
N. tabacum x N. alata.
1) F , of N. suaveolens (n=16)xN. longiflora (n=10)
At MI of PMC's of this hybrid, the number of bivalents ranged fromo to 4, with the mode at 1. The frequency of PMC's with 0 bivalents
followed that of PMC's with 1 univalent. PMC's with 2 and 3 bivalents
were frequently found but those with 4 bivalents were very rare.
In the same hybrid, Kostoff (1943) found that the number of bivalents
ranged most frequently from 0 to 3, and GOODSPEED (1954) also found therange from 0 to 4, although PMC's with 4 bivalents were very rare, but
CHRISTOFF (1929) observed 26 univalents and no bivalent in the same hybrid.
As KOSTOFF (1943) reported, quadrivalents are occasionally found.
Accordingly, it is difficult to speculate on the relationship between N.
suaveolens which is an Australasian species and N. longiflora which is an
American species, although these two species are much alike in external
characters.
2) F , of N. suaveolens (n=16)xN. plumbaginifolia (n=10)
At MI of PMC's of this hybrid, the number of bivalents ranged fromo to 2, with the mode at O. The frequency of PMC's with one bivalentfollowed that of PMC's without bivalents. PMC's with 2 bivalents were rare.
In the same hybrid, KOSTOFF (1943) found that the number of bivalents
ranged most frequently from 0 to 4, but CHROSTOFF (1929) observed 26 univalents and no bivalent in the same hybrid.
According to the above results, it is difficult to speculate on the relationship between N. suaveolens which is an Australasian species and N.
plumbaginifolia which is an American species, although the resemblancein their external appearance is remarkable.
3) F 1 of 4x N. tabacum (n=48) x N. alata (n=9)
This hybrid was produced for the purpose of introducing a gene or genes
for immunity against mildew and black shank, and for resistance against
wild fire and anthracnose from N. alata into cultivated tobacco.
At MI in PMC's of this hybrid, the total number of bivalents and trivalents per cell was 24. The number of trivalents most frequently ranged
from 1 to 4 but 0, 5 and 6 were rare.
At meiosis of the F 1 of N. tabacum x N. alata, the author (1956) observed 1-6 bivalents and the most frequent configulations found were
3n + 171 and 4n + 151. According to KOSTOFF'S description (1943), the same

64

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

hybrid N. tacacumxN. alata contained 5-9 bivalents and also some polyvalents. Further, the number of trivalents in the hybrid, 4x N. tabacumxN. alata, generally agreed with that of bivalents in the hybrid, N.
tabacum x N. alata, observed by myself, but did not agree with KOSTOFF'S
findings.
At AI of PMC's, chromosome bridges and strayed chromosomes outside the spindle were frequently seen. At 2nd division, they form small
nuclei and are the cause of polyspory at tetrad stage.

59.

Developmental studies in the genus Oryza II
Further studies on embryo sac formation(By Yukio Do IDA)
Table 1.

Materials and their origin

Species

Strain number

OriginO. sativa var. spontanea I

WOl06
W0120
W0002
W00l2

Phylankara, Cuttack, India
Cuttack, Orissa, India
BangkokCentral Rice Res. Inst., Cuttack, India(C. R. R. I.)
C. R. R. I.
Crop. Res. Div., USDA, Beltsville, Maryland, U.S.A.
C. R. R. I.
C. R. R. I.
C. R. R. I.
Coimbatore, Madras, India
Chaco

O. perennis

I

O. officinalis.
I

O. latifolia

W0019

O. minuta

W0045

O. eichingeri
O. australiensis
O. malabarensis
O. granulata
O. subulata

WOOl 5
WOOOS
W0021
W0004
W0510

--~---~-,------------In the previous report, the process of embryo sac formation was described for five Oryza species; O. sativa, O. glaberrima, O. alta, O.
breviligulata and O. ridleyi.
Similar observations were carried out in ten other species listed in Table
1. No conspicuous differencies in the process were found. Eight nuclei
were observed in the embryo sacs. Three formed the egg apparatus, twobecame polar nuclei and the remaining three were the antipodal nuclei.
After fertilization an abortive development was observed of the antipodal
cells which became multinucleate or polyploid and later degenerated.

65

RESEARCHES CARRIED OUT IN 1960

60.

Cytological studies in the genus Polygonum II(By Yukio DOIDA)

Cytological studies have been continued with the view of discussing the
problem of phylogeny of the Polygonaceae family.
The results are summarized in Table 1.
P. debile has sixteen chromosomes in root-tip cells. The chromosomes
are markedly larger than those of other species. The basic chromosome
number of this diploid species is 8. This number considered to be basic
Table 1.

- ---- -Chromosome numbers of the Polygonum species examined---------- -- - i

Species name

Polygonum debile Meisn.
P. weyrichii Fr. Schm.
P. senticosum (Meisn.)Franch et Savat.
P. nepalense Meisn.
P. bistorta Linn.
P. bistorta Linn.
]:*:

Chromosome
number (2n)
16
20

,
I

Previous
reporters

Localities

Su:

Nikko
Nikko*

20

Nagoya

24
48
(24)
48

]:

44

So:

46

Nikko
Mt.lbuki
Nikko*SOKOLOVSKAJA et al. (1938), Su: SUGIURA (1925)
Supplied by Nikko Botanic Garden of Tokyo University,]ARETZKY (1928), So:

for the genus was found among other Polygonaceae only in the genera
Fagopyrum and Rumex. The size of chromosomes in both, however, isconsiderably smaller than those of P. debile. The number n=8 was reported for the first time for a Polygonum species.
Pollen grains of P. debile belong to the" pore type" but those of Rumex
and Fagopyrum species are of "furrow type". The relationship between
P. debile and the two genera, Fagoyprum and Rumex, is considered to berather distant.
P. bistorta was obtained from the Nikko Botanic Garden of Tokyo University. Fourty eight chromosomes were counted. Samples of the same
species collected from Mt. Ibuki at Shiga-ken had twenty four chromosomes (d. Ann. Rep. Nat. Inst. Genet., No. 10).

66

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

61. Developmental studies in the genus Polygonum IV
Further studies on the effect of gibberellin on microsporogenesis
in three Polygonum species(By Yukio DOIDA)The effects of gibberellin (GB) on the microsporogenesis of Polygonum
jagopyrum was reported in the previous paper (Ann. Rep. No.9), in which
the range of gibberellin concentration used was 0, 0.1, 1, 10, 25, 50 and
100 p.p.m. The number of pollen grains was not strikingly altered by
the lower concentrations, but was strongly reduced by the treatment withsolutions of higher concentration than 10 p.p.m.
The effects of GB were further studied in 1960 with three Polygonum
species: P. jagopyrum (buckwheat), P. nodosum and P. blumei.
The solutions used were 0, 0.01, 0.1, 1, 10, 25 and 100 p.p.m. whichwere sprayed on whole plants. Buckwheat was treated from seedling toflowering stage at about 3 day intervals. The other two species werecollected in the state of nature and transplanted to pots May 17th. The
treatment was applied at 5 day intervals from May 18th to July 25th.
P
50

t3~Opjn..:m

o

..0

0

•

0

fI

P blume;

~
o

0

. ..

P nodosum

15• Aug 29oJuly 22

Aug. 29
10

0

z 5

20

July 2215cc

8 E ;; ~ ~ §Cone. 0f GB (pp,n:,\Fig. 1

T
;:>

0

c

-

c

Cone of GB (p,p,m.1

Fig. 2

Fig. 3

Inflorescences were collected July 22nd and August 29th. P. blumei
flowered July 22nd except for the plants which were treated with 100
p.p.m. solution, while on the same day of P. nodosum only plants treatedwith 100 p.p.m. flowered.
The results obtained as to the number of pollen grains are given in
Figs. 1-3. The figures show a similar behavior for 2 species, P. jagopyrum and P. blumei. It was ascertained that the number of pollen grains
increased by the treatment with 0.01 p.p.m. Other concentrations showedno effect or an inhibiting one. No effect of GB was observed for all con-

RESEARCHES CARRIED OUT IN 1960

67

centrations Aug. 29th. All previously affected plants have recovered theability to form the normal number of pollen grains.
The effects on the vegetative organs such as plant height, number ofinternodes and leaf size were also investigated. Plant height was increased at higher concentrations.
It may be concluded that GB stimulates cell divisions within a certain
concentration range, effectiveness being different in vegetative and reproductive organs of the examined species.

62.

Studies on flower color variation due to anthocyanins(By Toru ENDO)

Cellular factors of flower color variation due to anthocyanins have been
investigated. The present results were obtained by determination of themain component of anthocyanin, estimation of Iso/L of aqueous extracts
frrm fresh (and/or dry) flower petals with distilled water and comparisonof absorption maxima of crude extracts and of pure anthocyanins in both
1% aqueous and 0.01% methanolic hydrochloric acid solutions. The latterprocedure was employed to detect metal complex and co-pigmented anthocyanins. Some of the results are shown in Table 1, in which thedegree of stability of the aqueous extracts is estimated by the agreement
between the flower color and the color of the aqueous extract 30 min.
after extraction, besides value of 130/11. 11 means optical density of theaqueous extract immediately after extraction from flower petals and 130
optical density 30 min. after extraction. For the estimation of 130/11, 530,
560 and 580 m,u were used for reddish, purplish and bluish aqueous extracts, respectively.
As shown in Table 1. flower colorations can be apparently classified
into two groups, stable and labile, according to the degree of stability of
the aqueous extract. It is evident that the classification is not related
either to the kind of the main anthocyanin component or the original
flower color. Thus, it may be concluded that one of the m3.in factors inflower color variation due to anthocyanins is based on the cellular colloidal system, which is basically composed of cell-sap soluble high molecular
substances such as polysaccharides, proteins, polypeptides and someothers, most of them being qualitatively and quantitatively different according to species or strain and strongly or weakly combined with theanthocyanins.

en

00

>
Z
Table l.

Flower color

Plant nameZ

Stability of colors in aqueous extracts from flowers of several plants
Color ofaqueousextract

Main anthocyanidin

c:::::>
r
Iso/I!

Stability

Note

'"0

0

?:l
>-3

Reddish purple

CyanidinRedo (browing)

Pansy

Deep purple

Delphinidin

Purple

0.61

Pansy

Purplish blue

Delphinidin

Blue

0.74

Comparatively
Co-pigment
stableI

Torenia

Deep purple

MalvidinRed--

LabileDahliaRed

CyanidinRed

LabileMorning glory

Bluish purple

PeonidinRedo (browing)--

LabileAnchusaBlue

Petunidin

Gray

--

LabileCommelina

Blue

Delphinidin

Blue

0.97

StableRose

Red

Cyanidin

Red

0.96

StableCornflower

Blue

Cyanidin

Blue

0.90

Stable

Pansy

I

!

?:l
t'l

I

Labile0"'lComparatively
stable

Z

>
>-3

.....
0
Z

r>

Z
U1

>-3
.....
>-3
c:::::>-3t'l

Metal complex

0

"'l

C'l
t'l

Z

Metal complex

t'l

>-3

n
U1
z
9

......

......

RESEARCHES CARRIED OUT IN 1960

63.

69

Anti-cancer activity of Gentiana extract(By YO TAKENAKA, Yoshito OGAWA, Toru ODASHIRO and Noriko KARUBE)

We reported that the extract from Gentiana scabra Bunge var. buergeri
MAXIMOWITZ showed remarkable radiomimetic effects upon the living rootsof Allium scurodoprasum var. l'iviparum and Vicia faba. This finding
suggested the possibility of applying it with good results in the treatmentof tumors, because Gentiana scabra var. buergeri is not poisonous to humanbody. The effect of the extract on Yoshida sarcoma, transplanted into astrain of rat, Wister (two months old), is reported in this paper.
Gentiana Radix Pulverata was treated with methanol. After evaporation of the solvent in vacuum at 50°C., a dark brown oil residue remained.
Immediately after the transplantation of the ascites tumor, the obtained
extract (0.5 mg. per 1 g body weight of rat) was injected at the axilla
subcutaneously, and its influence on the sarcoma was observed in respect
to the following three points: 1) Mitotic activity in sarcoma cells. 2)
Systemic symptoms of the rat bearing the sarcoma (appetite, ascites,
icterus and metastasis). 3) Prolongation of the host's life.
In the mitotic activity of sarcoma cells, a temporary decrease (significant et 5% level) was observed 48 hours after injection of the extract.
But no significant difference was recognized between the treated groupand the control in the frequency of dividing cells three days after transplantation. As to the systemic symptoms of the host rat, no difference
was found between the treated and the control groups in the first fivedays, but seven days after injection, no ascites was found in the abdomenof the treated rat 15 days after injection, the treated group showed aremarkable recovery in systemic symptoms and 60% of the treated animals
remained alive for more than 70 days after transplantation of the sarcoma(Fig. 1.), though the control group;afterc trnnsplaotation
died out between the 10th and. . ""1__
the 15th day. No toxicity of the::: SOc
Treat
extract applied in the above menClimcal stage
tioned doses was oberved, except
COl
Coat.
for some inflammation at the inFig. 1. The effect of Gentiana extract
jected site observed only in theon life-prolongation of the host.
first three days after treatment.
These experimental results seem to indicate that the effect of the
Gentiana extract on sarcoma cells may be not direct but due to a secondary metabolite. Chemical the pharmacological research of this effective
Gentiana component is now on the way.
II;,}S

1l(J)

t ....

\10,-~0__ ~~_~~ __~_: ~O

70

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

C.
64.

MATHEMATICAL GENETICS

An example of genetic system in which the gene frequency
equilibrium is stable under negative overdominance(By Motoo KIMURA)

Let Xl and xz( =l-xl) be respectively the frequencies of a pair of alleles
Al and A z in a random mating population and assume that the relative
fitnesses of the three genotypes AlAI, AlA z and AzAz are expressed bylinear functions of gene frequencies as follows:
GenotypeoRelative fitness-Sl(Xl - Cl)(Malthusian parameters)

Here, we assume that Sl, Sz, Cl and Cz are all positive and constant. Sucha situation may be realized if the environment consists of two different
niches, el and ez, in which AlAI is adapted to the former (el) and AzA z
to the latter (ez), while the heterozygote AlA z is adapted uniformly to both.
The average fitness of the population isand the gene frequency equilibrium is attained when

In order to simplify the situation, let us assume that Sl =S2==S( >0). Thenthe equilibrium frequency of Al is
1-cz
A

Xl=

.

2-Cl-CZ

The necessary and sufficient condition for the equilibrium to be stable
and non trivial is that both Cl and cz lie between 0 and 1, i.e.
0<C1<1,

O<Cz<l.

Relative fitnesses of AlAI, AlAz and AzAz at equilibrium are,-s(l-cl-cz)(l-cl)al1=---2-Cl-CZRESEARCHES CARRIED OUT IN 1960

71

Here we may distinguish two cases:
Case 1. 0<Cl+C2~1. In this case, 1111<0 and 1122<0, so that the heterozygote A l A 2 is advantageous over both homozygotes.
Case 2. 1<cl+c2<2. In this case, 1111>0 and 1122>0, so that AlA2 isless advantageous than either homozygote (negative overdominance). It
is interesting to note that in this case, the gene frequency equilibrium isstable under" negative heterosis".
In both cases, the equilibrium frequency of Al is generally different fromthe frequency in which the average fitness of the population is maximized, i.e.
(Xl )max=

65.

3-2C2
6-2cl-2c2

Mutational load when the phenotYPic eifeet of mutational damage
is partially suppressed by developmental homeostasis(By Motoo KIMURA)

We will consider a random mating population of a diploid organism .
No restriction will be made to the number of segregating loci except thatthe average number of mutant genes per individual is assumed to besmall. Namely, it will be assumed that the frequency of mutant genes
per locus is sufficiently low so that only mutant heterozygotes are required to be considered for the calculation of mutational load.
Let D be the total mutational damage per zygote measured in lethal
equivalent (d. Morton, Crow and Muller 1956). If the frequency ofmutant gene in the kth locus is Slk) and its selection coefficient is -Slk),
then the average mutational damage may be expressed by
D = ~ 2X lk )Slk) ,kwhere summation is over all relevant loci.
cies are given by

The equilibrium gene frequen-'Ik)=~X(:La)'
dx lk )

where p.1};) is the mutation rate in the kth locus, a IS the average fitnessof the population in which the fitness, measured in Malthusian parameters,of the normal genotype is taken as standard (0) and the" hat" ("") on aletter designates that it is the value at equilibrium.

72

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

If there is no epistasis, fitness of individual genotype is proportional togenetic damage, i.e.

a=cD,
where c is a positive constant (here corresponding to the degree of dominance of mutant genes in fitness). In this case it can easily be shownthat the mutational load is equal to the total mutation rate per zygote:

LJL=ldl = ~ 2fJ-Ik) •k

A simple but an interesting situation will be produced by assuming that
fitness of an individual genotype is proportional to the square of genetic
damage, i.e.

a=-cD2.
Then the mutational load is given by

where M is the total mutation rate per zygote, i.e.
M= 22 2fJ-Ikl
kand 15 is determined by the relation

As an example, let s=1 for all loci (that is, all mutations are lethal), M=0.4 and c=0.05, then we have

Similar calculations may also be extended to the case of stronger epistasis such as
a=-cD3.

66.

Population dynamics of selfing(By Terumi MUKAI)

Since the contributions of males to the fitness of a genotype are usually different from those of the females, it stands to reason that the fitness
may not be a simple function of its component factors in random mating

RESEARCHES CARRIED OUT IN 1960

73

populations. This should also hold true for selfing populations. Therefore,a study was carried out with respect to a single locus in order to clarify
the effect of each fitness component on the relative frequencies of the
genotypes.
In order to set up a mathematical model, the following parameters
were defined: A and a=genes in the locus in question. m=relative pollen competitive ability of a-carrying pollen grains, that of A-carrying
ones being 1. sl(s2)=relative seed productive ability of Aa individuals (aaindividuals), that of AA being 1. vI(v2)=relative zygotic viability of Aa
individuals (aa individuals), that of AA being 1 (germination percentage
and selection before maturation). n=generation number.
The relative frequencies for mature plants (AA, Aa and aa) in F ngeneration can be expressed as follows:fFn(AA)fF'n(Aa)

1
L+MvI+N

V2

MVI
L+MvI+Nv2

(1)

..
)
MV2
/F'n(aa =L+ MVI +NV2

where L=(SIVI-2s2V2)[2n- I -(SIVI)n-IJ, M=(l +m)(2-slvI) X (SIVI-2s2V2)(SIVI)n-2
and N=m(2-slvI)[(SIVI)n-I_(2s2V2)n-I]. (SIVI=F2 and SIVI=F2s2V2).
The relative frequencies for seeds for F n generation can be easily obtained by equating VI =V2 = 1 in Formula (1) except for v, and V2 in L, M
and N.
Under the conditions of S,V, >2 and S,V, >2S2V2, the population reaches
a stable equilibrium. The equilibrium frequency can be obtained by taking the limiting values of the formula given above when n is approaching
infinity. Details of this work have been published in Seiken Jiho 11.

D.
67.

GENETICS AND BIOCHEMISTRY OF
MICROORGANISMS

Transduetions between curly flagellar mutants in Salmonella I)(By Tetsuo IINO)

In order to obtain recombinations between curly flagellar determinants
in Salmonella (Annual Report, No.9, 1959) transduction was carried out1) This work was supported by a research grant from the National Instituteof Allergy and Infectious Disease (E-2872), Public Health Service, U.S.A.

74

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

from curly i-phase of SW577 to curly a-phase of SL23, and from curly
enx-phase of SJ30 to curly 1.2-phase of either S]167 or S]168. SW577 is
a mutant obtained from a diphasic strain of S. typhimurium R-14. SJ30,
S]167 and S]168 were obtained from a phase-2 stable strain of S. abortusequi, SL23, and its transductional derivative SJ25 (stable 1.2-type). In thefirst combination of the transduction, a phase-2 culture was used for the
preparation of phage-PLT22 lysate and the transduction of Hzl.z was scored as control: when a phase-2 culture is used as a donor transduction of
HI and Hz are expected to be equal. In transductions from SJ30, SL23
was used as the donor in control experiments.
So far, the normal flagella type which carry antigen type of the donor
has not been obtained from any combinations, while in each control combination over 500 transductional recombinants were obtained.
It suggests that the mutant sites of these curly types are very closely
linked or identical. As demonstrated by Kerridge (1959), curly flagellaare produced when phenyl-alanine is. replaced by p-fluoro phenyl alanine
in flagellar regeneration. Therefore it might be quite possible that the
curly flagellar mutants were produced by replacement of a certain aminoacid in flagellin by the other; such mutants might occur by mutation ofa specific site in HI or Hz.
In a combination of the control experiments, in which SL23 was used
as a donor and S]168 as a recipient, 126 normal flagellar type clones with
enx-antigen were detected. Among them, two clones were found to be
agglutinated by anti-1.2 serum as well as enti-enx serum. They were inhibited spreading on nutrient gelatine-semisolid plates by either anti-enx
serum or anti-1.2 serum. Their segregation to the pure enx- or 1.2-type
cells in successive subcultures has not been observed. In these two clones
recombination might have occurred in an antigen type determinant Hz
coupled with the flagellar shape determinant. Agglutination tests withsingle factor antisera on these recombinants are in progress.

68.

A non-flagellated mutant which produces flagellar protein
in Salmonella l )(By Tetsuo IINO and Ichiro HARUNAZ)

S. abortus-equi SJ28 is a non-motile mutant obtained from a phase-2(enx-type) stable strain SL23. The mutant does not produce flagella andis not agglutinated by anti-H (enx) serum. The mutant character of1) This work was supported by a research grant from the National Instituteof Allergy and Infectious Disease (E-2872), Public Health Service, U.S.A.
2) Virus Research Institute, Kyoto University.

RESEARCHES CARRIED OUT IN 196075

SJ28 is very stable; so far, the reversion to a motile tipe has not been
observed.
Antiserum was prepared against a 0.5% formaline-saline suspension of
SJ28 cells, which contained 5 x 10 6 cells per ml: 0.5 to 2.0 ml of the suspension was injected intravenously to a rabbit seven times at 3 or 4 days'
interval. The anti-SJ28 serum thus obtained showed both H- and O-agglutinations when it was mixed with the cells of SL23. After complete absorption of the anti-SJ28 serum by SL25, which is an 1.2-type transductional derivative of SL23, and by heated SL23, an enx-specific antiserum
fraction (tube agglutination titer: 16,000) remained. The specificity of
the anti-enx serum fraction was further confirmed by a precipitation testwith isolated flagellar proteins of different antigen types.
The chromatographic fraction (fraction-f) which corresponds to flagellar
protein was obtained from SJ28 by 'sonication-DEAE method'. Though
the amount of protein in the fraction-e from SJ28 is less than one twentieth of that from SL23, the presence of enx-specific antigens in it wasdemonstrated by precipitation tests. Furthermore, on a rabbit immunized by the fraction-e of SJ28, the production of anti-enx serum of titer8,000 was observed.
From these results, it is inferred that SJ28 is a mutant which can
synthesize protein with the same antigenicity as a flagellar protein of theparent strain SL23, but the mutant cannot organize such protein molecules into a flagellum. A part of the enx-type protein produced by SJ28
may leak out from the cells.
In contrast with SJ28, proteins with H-antigenicity have not been detected from a deletion type of non-flagellated (Fla-) mutant SJ157.

69.

Host-range mutants of chi-phage!)(By Hi ro SASAKI)

It has been known that chi-phage attacks Salmonella having flagellar
antigen with the exception of the following two distinct kinds of flagellated types: one which has flagella but is not motile (so-called paralysedtype), and the other which has g-antigenic flagella. In the latter type,the phage resistance is generally antigenic phase specific.
As a new type of chi-resistant bacteria, motile bacteria resistant inboth flagellar antigenic phases were obtained from S. panama (Iv: 1,5),
S. lomita (eh: 1,5), S. reading (eh: 1,5).
1) This work was supported by a research grant from the National Institute ofAllergy and Infectious Diseases (E-2872), Public Health Service, U.S.A.

: 76

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

Three phage types which can attack chi-resistant SJ 26 (gp: (1,2)), SJ
181 (Iv: 1,5), and SJ 8 (mt:) were obtained from chi-phage developed inserial mixed cultures of phage-sensitive strain, SL 23 (a: enx) and eachof the resistant bacteria strains. It was proved that these phages are indistinguishable from original chi-phage in serological specificity, hostspecificity (: they attack flagellated bacteria but not non-flagellated bacteria),
growth characteristics on the common host SL 23, and electron-microscopic morphology of their particles. These phage types originated from SJ
26, SJ 191, and SJ 8 were designated h1, h2, h3, respectively.
Host-range and efficiency of plating are approximately the same between
h1 and h3 on most of the bacterial strains having g-type flagella. They
can not attack SJ 181. In contrast with h1 and h3, h2 attack SJ 181 but
for cannot attack g-group bacteria. Phage-h1 propagated through SL 23
can grow in every cell of SL 23 but only in about one of 10 6 cells of SJ
26 (gp:(1,2)). The same phage-h1 propagated through SJ 26 (gp:(1,2)) cangrow in all cells of SL 23 and in about one of 10 cells of SJ 26 (gp:(1,2)).
However, after its propagation through SL 23, its infectivity to SJ 26 isreduced again to one in 106 cells. Thus, a remarkable host controlled
variation by SL 23 and SJ 26 was observed on hI. On the contrary, h3does not show the variation. When a suspension of h3 is plated with SJ
26 (gp:(1,2)), it produces a smaller number of plaques than with SL 23.

70.

The composition of ribonucleic acid in Yeast irradiatedwith ultraviolet light(By Saburo NAWA)

The nucleotide composition of ribonucleic acid synthesized in the irradiated cells of yeast was determined. After the incubation of UV irradiated cells in the minimal medium containing 32p (orthophosphate) for
30 min., RNA was extracted using the Davidson and Smellie procedureand digested with potassium hydroxide. The nucleotides were chromatographically separated using a column of Dowex 1-8x formate and theactivity of 32p of each nucleotide was measured.
The nucleotide composition of the RNA fraction from the irradiatedcells showed a higher value of CjA(C+CjA+U) and a similar value of
CjG or UjA as comparied with RNA from non-irradiated cells. It wasobserved that the composition of RNA synthesized in irradiated cells wasaffected by the supplementation of certain nucleic acid precursors to theirradiated medium. The ratio CjA decreased when adenosine and guanosine were in combination supplied to irradiated cells, although additionof ribonucleotides had no effect when they were singly supplied. It is

RESEARCHES CARRIED OUT IN 196077

noticeable that addition of deoxyribosides could modify the composition of
RNA in irradiated cells. Supplementation of deoxyadenosine plus thymidine let to normalization of the ratio CIA. This suggests that the synthesis of the abnormal RNA in irradiated cells involves some turnover ofDNA. No effect was observed of above supplementation on the nucleotide composition of RNA in non-irradiated cells.

71.

Nuclear structure of yeast(By Yoshiaki YONEDA)

The nuclear structure and the division process of yeast were investigated. The materials used were haploid, diploid and tetraploid strains of
the Carbondale stocks of Saccharomyces.
As previously reported, the extravacuolar nucleus of haplo-, diplo- andtetraploid strains is composed of two parts; one is the Feulgen-positive
chromatin region and the other is the Feulgen-negative karyoplasm. Acetoorcein staining revealed that the reddish stained chromatin is situated on
one side of th~ nucleus which is attached to the central vacuole. At thenuclear division stage, the chromatin divides at first into two bodies,
whereupon the nucleus becomes constricted.
Later, the detailed structure of the chromatin region was observed bystaining with Feulgen and Giemsa methods; it could be also clearly observed with Feulgen and aceto-orcein combined staining. In the centerof the chromatin region, a small granule was found, which was Feulgenpositive and tentatively termed as central granule. A ring of homogeneous chromatin surrounded this granule at the resting stage.
At the first stage of the division, some parts of the chromatin ringchanged to condensed structures, from which chromosomal bodies originated in later stages. The chromatin condensation was remarkable throughthe last stage of the non-budding phase and during bud sprouting. Before a bud grew in length to one third of the mother cell, metaphase
chromosomal bodies appeared and anaphase was carried out. The number of the metaphase chromosomal bodies was not proportional to ploidy.
About three such bodies were distinguished in all haplo-, diplo- and tetraploid strains and at anaphase six or more bodies were observed. Simultaneously, the division of the central granule took place. When the bud
increased to half the size of the mother cell, the chromatin was completely separated into two daughter chromatin bodies. Thereafter, the elongation and constriction of the whole nucleus occurred in the mother cell or1) This work was supported partly by Grant RF 57178 from The Rockefeller
Foundation.

78

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

between it and the bud. The daughter chromatin body, moving to thebud, was compact and after settling in the bud, assumed a diffused appearance.
Recently, YUASA and LINDEGREN (1959) reported the existence of" centrioles" in yeast nuclei, the number of which corresponded to theploidy of the cell. The central granule may be comparable to those" centrioles" in regard to its position, though only one granule was foundin the strains studied.

72.

The effect of nucleoside supplementation on mutation!)(By Sayaka NAKAI)

Previous studies of the author in yeast and of Doudney and Haas in E.

coli have shown that there are several steps in mutation which are correlated with macromolecular synthesis after irradiation. The present experiments are concerned with nucleic acid base requirements in the mutative steps after UV irradiation by metabolite supplementation techniques
for the examination of molecular properties of UV induced mutations.
The mutation studied is a reversion of the isoleucine-requiring to the
non-requiring allele (il-~il+) in the haploid strain A9 of Saccharomyces
cerevisiae. In order to conduct this study, various nucleosides (50,ugjml),
singly or combined, were supplemented to the medium at 30 minute intervals from 0 to 3 hours after irradiation. The results obtained aresummarized as follows:
1) Supplementation of pyrimidine, especially, thymine deoxynucleosideimmediately after UV irradiation increases the surviving fraction (about20%), but deoxypurine nucleoside supplement has no such effect.
On the other hand supplementation of purine ribonucleoside increases
the surviving fraction (about 20%), but not pyrimidine nucleoside.
2) Purine deoxynucleoside supplement, especially, in combination of
deoxyadenosine and deoxyguanosine remarkably affects the mutation
frequency, changing it to a bimodal pattern as a function of time intervals
between irradiation and supplementation of nucleoside. The first peak
appears at 30 minutes and the second peak at 120 minutes after additionof the above compounds. On the contrary, pyrimidine deoxynucleosidesupplement does not show such relationship.
Effect of ribonucleoside supplementation on mutation frequency is somewhat different from that of deoxynucleoside supplementation. In general,pyrimidine ribonucleoside has a similar effect to that of purine deoxynucleoside.
1) This work was supported partly by Grant RF 57178 from The Rockefeller
Foundation.

RESEARCHES CARRIED OUT IN 1960

79

The results lead to the following hypothesis: Genetic damage caused
by UV irradiation primarily occurs more frequently at the site of pyrimidines in DNA molecule. Supplemented deoxypurines may partly substitutefor the damaged pyrimidines in the process of mutation induction.

73.

Variations in the number of micronuclei found zn the wild
populations of Paramecium polycaryum(By Susumu MURAMATSU)

Paramecium polycaryum is commonly found in foul water containing
large amounts of organic substances, and is characterized by the numberof micronuclei. It has been known that the number of micronuclei varies
from one to eight, although four is the typical number.
The present report deals with the variation of micronuclei counts often wild populations of the species collected in Hakodate, Hokkaido. The
micronuclei counts in these samples varied in a wide range from one tothirteen. From Table I, it is found that the majority of animals prossess
three to five micronuclei. As compared with other populations, however,
HM, HG and HA populations which were collected in foul water without
any contamination by sea-water or hot-spa, exhibited a rather small
amount of variation in the micronuclei counts and had also a low incidenceof aberrant and polymicronucleate animals. On the other hand, the otherpopulations, HK, HN, HT, HS, HD, HY and HU which were collected in
the habitat considerably contaminated by sea-water or warm-water drainage from hot-spa, exhibited comparatively large range of variation in
the micronuclei counts, and had a high in cidevant of aberrant andpolymicronucleate animals.
From the above investigations, it seems that a certain relationshipexists between the numerical variation of the micronuclei and the environmental factors. At present, it seems that differences in water temperature and salinity are most important factors in the variability of thecounts.
The analysis is now in progress of the seasonal change of variability
within populations.

00
0

>
Z
Z

Table l.
rtJ'+-I~'"d , 1j*cn

Popula- I o.g ~.. ,...., H

tions

I

I Z'O.Di!5.i': ~I ,.sO

I

c:::

Number of Micronuclei

r:: ~8 I~ '8I ",;",;

I

1

2

3

4

5

6

7

8

%

%

;%

%

%

% -%---%-

5.2

9

10

11

%

%- %

12

13

---

-~

--

---

... t::

0.8

~~
<1J'~

o H

u~

--

%

0

~

>-l
0

'"!j

-

Z

0.236

-

3. 773±0. 993

0.263

1.2 0.2-

3. 733±0 .835

0.223

9.6 7.5 4.3 1.8 0.5 0.3 0.1-

4.231 ±1.897

0.448

7.6 14.5 16.3 18.6 13.7 12.8 10.9 3.1 0.5 0.2 0.1-

4.285 ± 1. 785

0.416

2.0

7.7 11.3 18.1 15.0 15.6 12.2 7.9 5.1 2.7 1.8 0.3 0.1

0.2

4.515±2.256

0.499

1150

0.4

6.8 16.3 15.6 19.3 16.5

9.8 8.3 4.9 1.2 0.4 0.2 0.2

0.1

4.243±2.045

0.481

1895

3.4

8.6 14.8 14.5 14.3 13.8 10.5 7.9 5.7 3.6 2.0 0.4 0.3

0.2

4.502±2.419

0.537

1892

1.3

8.4 23.2 14.7 30.1 12.6

3.467 ±1.453

0.419

Z

4.570± 1. 764

0.385

>-l

2049

0.2 I 1.4

7.3 76.7

4.2

2.1

HG

1908

0.1 I 1.5 10.8 12.3 67.2

3.1

2.8 2.1 0.1

HA

1250

0.2

2.0

11.7 69.3

6.3

HK

1761

1.6

9.3 16.6 14.1 19.8 14.7

HN

1372

1.7

HT

2381

HS
HD

9.1

2.0 0.5 0.2 0.01

9.5 0.2 0.1 0.03

-

I

-

-

I

HU

~

tTl
'"0

3.959±0.939

HM

HY

Mean valueofMicronuclei

l-<

>
t"""

2109

1.6 ! 2.8

8.1 24.3 11.0 14.1

26.6 9.8 0.6 0.8 0.1 0.1 0.04

0.1

* Aberrant animals involved are" ghost", "amicronucleate" and" amacronucleate" animals,

>
>-l
>-<

0
Z

>
t"""

Z
UJ

>-l
>-<
>-l

c:::

>-l
tTl

0

'"!j

CJ
tTl
tTl

n
UJ
Z
0
>-<
>-<

RESEARCHES CARRIED OUT IN 1960

E.
74.

81

RADIATION GENETICS IN ANIMALS

Two types of dose-rate dependence of radiation-induced mutation
rate in spermatogonia and oiJgonia of the silkworm!)(By Yataro TAZIMA, Sohei KONDO and Toshihiko SADO)

Studies on the dose-rate dependence of radiation induced mutation rate
have been carried out with silkworm spermatogonia and oogonia by
irradiating them with different dose-rates of X- and r-rays.
Both sexes of a wild type strain, Cl08, were exposed to X-rays or j
Cs-137 r-rays of high dose-rate or to Co-60 r-rays of low dose-rate at adefinite stage or during a definite period in early larval stages. The
dose-rate ratios between acute and chronic irradiation varied from 2500; 1
to 7600: 1, with the same total doses of 950r or 1000r. For the estimationof mutation rates we applied the specific loci method. The results obtained are as follows.
a) A type of dose-rate dependence consistent with the findings ofRussell, i.e., lower mutagenic effectiveness of chronic than of acuteirradiation, was found in the silkworm only in very early stages ofspermatogonia and oogonia, when the primordial germ cells are prevailingin the testis (Type I).
b) A new type of dose rate dependence, i.e., higher mutagenic effectiveness of chronic than acute irradiation was found in later stages ofspermatogonial development (Type II).
For an explanation of these apparently contradictory findings, thefollowing assumptions were made.
1) Primary mutation rates differ according to the stage of germ cells.
They may be highest at the stage of primordial germ cells, lowest at the
primary spermatogonial stage and intermediate at the secondary spermatogonial stage.
2) Repair of primary mutations as postulated by Russell for chronic
irradiation may take place in the germ cells with a long mitotic cycle but
is less frequent in those with a short mitotic cycle. Primary and secondary
spermatogonia are supposed to have a much shorter cell cycle than theprimordial germ cells. Hence, recovery is supposed to play an importantrole only in the primordial germ cells.
3) Among the three types of cells, the secondary spermatogonia, which
supposedly correspond to type B spermatogonia of mouse, are extraordinary
sensitive to radiation. They are selectively killed by intense dose rates,
1)

This work was supported by Grant RF57178 from The Rockefeller Foundation.

82

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

while primordial germ cells and primary spermatogonia can survive underacute dose rate conditions such as were used in our experiments. Underchronic irradiation all three types of cells can survive and are transmitted
to the next generation.
With these assumptions, both types of dose rate dependence could be
explained without contradiction.

75.

Frequency of accumulated recessive embryonic lethals in a popularcommercial race, ellS, of the silkworm and theirspontaneous mutation rate l )(By Yataro TAZIMA and Takao KOBAYASHI)

An estimate of the frequency of accumulated recessive embryonic lethals
was made in a highly popular commercial race, C1l5, of the silkworm.
The materials used were the original PI eggs of this race, which were
produced for distribution purpose by the National Sericultural Experiment
Station in 1959. 200 PI layings, selected at random, were raised separately
in the spring of 1960. By sib-mating within the same PI separate batch,
P:;{l958)

P2(l95::J\

Fl(1960-!)

GZ('60-3)

Fig. 1. Breeding system utilized for extractionof lethal free strains and estimation of spontaneous lethal mutation rate.

about 100 layings of GI generation were obtained for each batch. They
were subjected to an embryonic lethal test, after treatment with hothydrochloric acid to induce hatching. Within each PI line, the numberof GI layings with about 25%, 43.8% or more dead embryos was scored.
From the ratio of lethal segregating layings to totallayings, the numberof recessive lethals carried by the grandparents (P2) was estimated. The
1) A part of the work carried out under the Contract No. 28 between I.A.E.A.
and Dept. of Morphological Genetics.

83

RESEARCHES CARRIED OUT IN 1960

results are shown in Table l.
From the data the frequency of lethals that had been transmitted from
Ps to P 2 were calculated. Since the number of gametes produced by P 3
and transmitted to both parents (P2) of each P, batch are four, the frequency of accumulated lethals (In) per gamete produced by P 3 is
L=0.6011/4=0.15026
Table 1.

Detection of accumlated recessive embryonic lethals in C115
Number of P , lines

Total
tested-----------

188

0
97

Detected-lethals per line2
3
1
75

16

2

------

Total No.
of lethals
detected

Freq. oflethals per
P , line-------------

113,--------

0.6011---------

This figure may also be taken as representing the frequency of spontaneous
lethals accumulated and maintained, in every generation, in a large population of this race.
Among 95 lethal free lines, a few lethal free strains were extracted.
With one of those, an experiment was carried out to estimate the spontaneous lethal mutation rate.
By random mating of G , moths of this strain, G2 layings were raised
separately. By sib-mating within each G2 batch, G3 layings were obtained
and they were tested for their embryonic lethality by the same procedure
as described above.
Among 309 G2 lines observed 10 were lethal yielding. Therefore thefrequency of lethals per gametes transmitted to both parents of each G 2batch is calculated as
10 =0.008091
309x4
As all lethals accumutated down to P s generation had already been
eliminated by sib-mating of P" and lethals newly arisen in G, and G 2
could not have been detected in the present test, the observed lethaIs
must have occurred during P 2 and Pl.
The calculated mutation rate per gamete per generation is, therefore,
p.= 0.008091
2

0.004046

This may be regarded as an estimate of the spontaneous mutation rate-of recessive embryonic lethals in this race.

84

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

76.

Sensitivity and time of degeneration of spermatocytes irradiatedat different stages of meiosis of the ,silkworm1)(By Toshihiko SADO)

An extremely sensitive stage to the sterilizing effect of X-rays has been
revealed in the silkworm early in the fifth instar of the male larva.
Hypersensitivity of secondary spermatogonia (SADO 1959) cannot satisfactorily account for this, because at this stage most germ cells are alreadydifferentiated into spermatocytes.
Spermatocytes, especially during first meiotic prophase, represent a very
long-lived stage in the silkworm. The majority of germ cells in the testisof the early fourth instar larva are spermatocytes at synaptic and/or
pachytene stage, while in the early fifth instar most are in late meiotic
prophase. To investigate the effect of X-rays on the spermatocytes atdifferent developmental stages both early fourth and early fifth instar
larvae were exposed to X-rays of 1000r and 2000r.
Most of the spermatocytes irradiated with 1000r at an early meiotic
stage showed no degenerative figures at meiosis. Fertility tests showedthat they developed into functional sperm. However, those exposed to
2000r showed structural abnormalities and degenerated during the spermiogenic stage.
The majority of spermatocytes irradiated at late meiotic prophase withlOOOr were normal in appearance at metaphase and anaphase, but seemed
to be damaged, for spermatids with necrotic nuclei were observed frequently 5 days after exposure. Still later in the pupal period eupyrene
spermatozoa with structurally abnormal heads (nuclei) appeared in thetestes. Thus spermatocytes in late meiotic prophase are more sensitive
to irradiation than those in early meiotic prophase.
It is highly probable that the majority of the observed structural abnormalities occurred in cells that carried gross chromosomal disturbances.
Actually chromosome aberrations have been observed in some of theirradiated testes. However, the detection of such aberrations is not easyin the silkworm, because the chromosomes are too small and numerous(n=28) for cytological analysis.

77.

Reduction in number of spenn after irradiation of male silkworms 21(By Toshihiko SADO)

The foregoing experiment suggests that the number of sperm producedin the testes irradiated during the early fifth instar would be reduced.
1) This work was supported by Grant RF57178 from the Rockefeller Foundation.
2) This work was supported by the Grant-in-Aid from the Ministry of Education.

RESEARCHES CARRIED OUT IN 1960

85

A quantitative histological study was carried out to confirm this prediction
from counts of the numbers of eupyrene and apyrene sperm bundles in
the irradiated and control testes. Throughout the pupal period, the numberof bundles of apyrene spermatozoa was found to be greatly decreased in
the irradiated testes. This, however, cannot account for the observed
sterility of the treated males, because even in nonirradiated males apyrene
spermatozoa are not considered to playa role in fertilization.
In irradiated testes, the number of eupyrene sperm bundles on the third
day after pupation or later was about 50 per cent of that in the nonirradiated ones. Furthermore, about 30 per cent of the eupyrene sperm
bundles in the treated individuals had structurally abnormal nuclei.
Though a considerable amount of sperm was contained in the testes of
the irradiated male moths and most of them seemed to have been ejaculated
into the bursa copulatrix of the female, only very few of them could reachthe receptaculums eminis. This show that most of them are functionless.
In conclusion, the excessive sterility of males irradiated during the early
fifth instar can be explained by the fact that cells in late meiotic prophase arehighly sensitive to irradiation and either degenerate during spermiogenesis
or form spermatozoa many of which are non-functional.

78.

Differences between times of death of the F, after irradiationof odgonia or mature odcytes ll(By Yataro T AZIMA and Kimiharu ONIMARU)

Marked changes in radiation-induced mutation rates between the stagesof gametogenesis have been known in the silkworm both for visible recessive mutations and dominant lethals. The induced mutation are recovered
with relatively low frequency at spermatogonial and oogonial stages, while
the maximum frequencies are observed at the spermatid stage in the maleand in mature oocytes in the female. In mature oocytes the observed
mutation rate is several times as high as that in the oogonia.
Moreover it was found by the specific locus method with egg color
mutants that the induced rates rise linearly with increasing radiation
doses in oogonia, while they increase exponentially with doses when mature
oocytes are irradiated. This suggests that most of the mutants inducedin oogonia may be accompanied by gross chromosomal aberrations, andthat the aberration bearing cells are eliminated afterwards during the longcourse of gametogenesis. On the contrary, most of the aberrations induced
in mature oocytes can not eliminated during the short time from irradiation to fertilization. It is, however, likely that most of the aberration
1)

This work was supported by the Grant-in-Aid from the Ministry of Education.

86

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

bearing individuals die some time during early development. Earlier experiments had, in fact, shown that the F , from the irradiated spermatids
and irradiated mature oocytes shows lower viability than the F , from
irradiated spermatogonia and oogonia. In order to confirm this, mortality
was measured at various developmental stages of the F, of irradiatedoogonia and irradiated mature oocytes as in controls. Doses given were
1000r of Cs-137 r-rays. The experiment was carried out in three replications.
Table 1.

Mortality in F , of irradiated oogonia, irradiated mature
oocytes and non-irradiated control

Treatment

Non-irrad. ¥X non-irrad. -er;
2.* Irrad. oogonia ¥x non-irrad. "63.* Irrad. mature
oocytes ¥X non-irrad. "61.

No. ofzygotes----observ- i Emed
bryo
2064
1851
2213

i
I
I

Per cent died during
III
III V--S··--Pupation- - - -.
-.
pmn:ng- Emer- Total
StadIUm StadIUm PupatIOn
gence

1.1%

2.4%

2.3%

3.2%

2.4%

11.4%

2.3

4.1

2.2

5.2

2.0

15.8

21.4

9.0

6.1

5.8

2.5

44.8

-----,--~*

Irradiation dose:.'. ---_._---------

1000 r of Cs-137 ,-rays

The results were completely in accordance with our expectation: a considerable part of the F , individuals of irradiated mature oocytes were
found to be eliminated during embryonic and larval stages, especially inearlier stages.

79.

Recessive lethal mutations induced in mice by chronic irradiation
during the whole development in three successive generations(By Tsutomu SUGAHARA, Kiyosi TUTIKAWA, Susumu MURAMATsu,
and Yoshiko TAKEDA)

We studied by Haldane's method recessive lethal mutations in miceinduced by chronic irradiation of 80 day duration, starting immediately
after fertilization and continued through three successive generations.
The result obtained seems to indicate that the genetic effect of radiation
is cumulative, even at a very low dose rate.
Multiple recessive mice were irradiated chronically with gamma-rays of
total dose of about 103r for three successive generations. In each generation, the mice were raised until maturity in the radiation field, then

87

RESEARCHES CARRIED OUT IN 1960

mated by placing a pair in a cage at the end of the irradiation. To
search for recessive lethal mutations, the offspring from irradiated ancestries were mated with mice of a wild-type strain, and the segregation for
the recessive marker genes in the F 2 progenies was examined for each
Fl pair. The criteria used for scanning linked recessive lethals were thesame as those used by CARTER (1959) which were computed for the probability of false clue pzO.01.
The" wavy" stock and the CBA strain were used as the multiple recessive and the wild-type strain, respectively. The" wavy" stock which
is homozygous for six loci, a, b, C Ch , d, P and se, was built up in 1957 by
K. TUTIKAWA. However, only four out of six loci were used as markers
in the present experiment, because d-se and CC11_p are closely linked.
The mice were chronically exposed to 60Co-gamma-rays for 80 days pereach generation with the dose rate of 0.43r/day. The results obtained aresummarized in Table 1.
Table 1.
Irradiated74

Number of P, mice analysed
Number of suspected recessive lethals
Total length of map scanned

Non-irradiated
46

2

o

5112.688 cM

4553.172 cM

Mutation rate
Observed

6.8 X 1O-3/r/Total autosomes

Expected

3.3-6.6 X 10- 3/r/Total autosomes

Pilot experiment (1959)

4.3 X 1O-3/r/Total autosomes

The total length of linkage map for a male mouse was estimated fromthe cytological data to be about 1824 centimorgans (SLIZYNSKI, 1955). Hence,
the total map length scanned in the irradiation series, i.e. 5113 centimorgans, and that in the non-irradiated series, i.e. 4553 centimorgans,were equivalent to 2.8 and 2.5 autosome sets, respectively, provided thatthe length of the linkage map of the female mouse is the same as thatof the male. Two suspected linked lethals were found in the P 1 mice of
the irradiated series, and no lethals were found in those of the nonirradiated series.
The expected mutation rate given in Table 1 is based on the value
estimated by HALDANE (1956), and the difference in mutation frequencies
between males and females is neglected. Since, in the present experiment,
the gametes irradiated at various stages of germ cells must have contributed to fertilization, the expected mutation rate has to make allowance

88

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

for doubling Haldane's estimated value, taking account of the higher
mutation frequency in the postgonial cells.
The observed rate is in good agreement with the expected value.

so. Shift of secondary sex ratio among the progeny of chronically
irradiated male mice (F , generation)(By Kiyosi TUTIKAWA)

Reports that the secondary sex ratio in man is modified in the progenyof irradiated fathers led us to the examination of sex distribution in theprogeny obtained from the above experiment. According to Schull and
Neel, who summarized the available human evidences, irradiation of thefather leads to a preponderance of males in the progeny.
CSMF, mice used in the present experiment were produced by mating
C57BL/6 females with SM/Rr males. The males were chronically irradiatedwith 60Co-gamma-rays of total doses of 100r (dose rate 2r/day), and 400r
(does rate Sr/day) for 50 days. In order to explore the possibility of theinfluence of age, two sets of experiments were carried out in which twogroups of 30- and 60-day-old CSMF, males were irradiated and two months
later, after completion of irradiation, were mated with non-irradiatedfemales of the same F,.
In brief, for each dose and age groups, the sex ratio (fraction of males)at birth was as follows:
(1)

Or

(2)

100 r

fO-daY-Old
60-day-old

400r

rO-daY-Old
60-day-old(3)1103 mice, sex ratio, 0.5512
519 mice, sex ratio, 0.5241
484 mice, sex ratio, 0.5351
418 mice, sex ratio, 0.4928
501 mice, sex ratio, 0.4830

The two age groups given the same doses, did not differ significantly
from each other in the sex ratio. However, the low sex ratio of the exposedgroups did significantly differ from that of the controls. Statistical tests(X 2 ) showed significant heterogenity among the three dose groups (O.Ol<P<0.025).
Recently, KOHN (1960), showed for the CAF , males used in his experiment, that X-rays have no effect on the sex ratio as tested by linear
regression for absorbed doses in the range 0-720 rads.
In our experiment, although gamma-rays had no effect on the meanlitter size, birth weight and skeletal development of new born animals,
the sex ratio decreased linearly with the doses. An attempt to explain

RESEARCHES CARRIED OUT IN 196089

the result was made under the assumptions that (1) difference from 1: 1
sex ratio of the control is probably due to the difference in competitive
ability between X- and Y-carrying sperm, (2) the frequency of non-disjunction in gametogenesis could have been increased by irradiation. In
this case, the frequencies were calculated to be 4% for 100r, and 12% for400r respectively. However, some doubt remains as to whether such highrates can be induced in gametogenesis by irradiation.

81. A radiation genetical consideration concerning the genetic
structure of natural populations in Drosophila melanogaster(By Terumi MUKAI)

There are two main mutually contradictory hypotheses concerning thegenetic structure of natural populations, (DOBZHANSKY, 1955), i.e., the classical hypothesis based upon the superior viability of homozygotes over heterozygetes and the balance hypothesis founded on the superiority of heterozygotes over either homozygote.
If one accepts the classical hypothesis, he would not expect to find any
real difference between autosomes and sex chromosomes in polygenes
governing viability, that is, most of the loci would be occupied by dominant
adaptive genes in homozygous condition, and an inbreeding depression is
not expected.
If one accepts the balance hypothesis he can expect the accumulationof variability in heterozygous condition in autosomes while not in sex
chromosomes because of the hemizygosity in males. Therefore, for theautosomes inbreeding depression is indicated but none for the sex chromosomes.
A study on the heterozygous effects of irradiation-induced mutations
occurring in artificially extracted isogenic lines would bring a considerable
amount of information as to whether the classical or the balance hypothesis
is well-grounded. It is well known that most mutations are deleterious
in homozygous condition (BRIDGES and BREHME, 1944). If this is the rule,
it is impossible on the basis of the classical hypothesis to increase theviability of heterozygotes by the induction of mutations either in autosomes or in sex chromosomes. However, if one adheres to the balance
hypothesis, the results should be slightly different. In the sex chromosomes, the situation would be essentially the same as indicated by theclassical hypothesis. (Indeed, DOBZHANSKY, KRIMBAS and KRIMBAS (1960)
have reported a tendency for the supervital chromosomes in D. pseudoobscura
not to show heterosis when they are combined with chromosomes having
loboratory-origin dominant genes.) But, contrary to expectation under

90

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

the classical hypothesis, the heterozygotes due to induced mutations in
autosomes under the balance hypothesis would show heterosis in viability
in the genetic backgrounds where the subvital genes are accumulated.
The statistical analysis of unpublished experimental data (BURDICK, MUKAI
and KRAWINKEL) has brought the following conclusion: There are twokinds of loci in D. melanogaster, and the balance hypothesis is applicable
to one kind, the proportion of which is about two thirds. The classicalhypothesis holds true for the other one third of the loci. Thus, it is
impossible to make an alternative judgement but the frequency of lociwhere the balance hypothesis is valid is higher than that where theclassical hyphothesis holds true.

82.

Radiation-induced polygene mutation rate
(By Terumi MUKAI)

When the characteristics of the distributions of treated and control
individuals are employed, the proportion of heterotic and non-heterotic
radiation-induced polygene mutations in heterozygous condition can be
estimated under the following assumptions:(1) There is no epistasis. This is too bold, but not dangerous if thenumber of mutations exceeds one in an individual in only few cases.
(2) Each mutation has the same effect either in the positive or thenegative direction.
(3) The positive and negative mutations distribute independently as
Poisson distributions.
In order to set up a mathematical model, the following paramaters aredefined: a=heterozygous effect of each mutation. p(q)=mean number ofnegative (positive) mutations in one set of chromosomes.
From the differences of means, variances and the third moment about
the mean between the irradiated and the control group, the following
simultaneous equations can be obtained:a(q-p)=Aa 2(p+q)=B
a 3 (q-p)=C
where A, Band C stand for the differences of statistics between theirradiated and the control group, respectively.
The heterozygous effect of radiation-induced mutations (acute lOOr) on
the viability of the second and third chromosomes in D. melanogaster was
estimated, based upon the sample sizes, n=296 for the treated group,n=279 for the control (BURDICK, MUKAI and KRAWINKEL, unpublished).
On the basis of the estimates A=O.025000, B=O.009973 and C=O.000660,

91

RESEARCHES CARRIED OUT IN 1960

a, p and q were estimated to be a=0.16, P=O.l1 and q=0.27. If weassume that the number of loci is 3000, we can estimate polygene mutation rate to be 1.26 X10- 6 /locus/r. This is extremely high compared withso called major-gene mutation rates.
83.

Frequency of chromatid breaks at various doses of X- and r-raysl)(By Tosihide H. YOSIDA, Teiichiro TAKAHASHI, Yoshikazu MATANO
and Kazuhiko UTSUMI)YOSIDA and TAKAHASHI (1959) demonstrated that 30 minutes after Xirradiation chromatid or isochromatid breaks considered to be one hit
aberrations occurred at the highest frequency in all samples obtained
immediately after, 30 minutes and 8 to 72 hours after 250r irradiation.
In order to investigate the relation between the frequency of chromatid
breaks and dose, 250, 500 and 750r of X-rays and 1,000r of r-rays
Table 1. The number of chromatid breaks per cell at various dosesof X- and r-rays

Dose (r)--,-ray

X.rays

Source
250

500

750

1000

2.6

5.3

6.14

8.5

--

No. of breaks per cell----I--------------(ee 137 ) were applied to tumor bearing mice. r-rays were used for short
exposures with 1000r. As shown in the table the frequency of chromatid
breaks by X- and r-rays was increasing in proportion to the dose, irrespective of the radiation source.

84.

Effect of DNP (2,4-dinitrophenol) on the frequency of Xray
induced chromatid breakes in Ehrlich tumor cells 2 )(By Yoshikazu MATANO)

In the present study, the effect of DNP on the frequency of X-ray
induced chromosome aberrations was examined, using hyperdiploid Ehrlich
carcinoma cells (chrom. no. 44) of mice.
Doses of 100r, 250r and 500r were given to tumor-bearing mice, whichwere beld separately in plastic cylindrical vessels. 0.2 ml of 10- 2 M DNP
1) This work was supported by Grant RF57178 from The Rockefeller Found:'\tion.
2) This work was supported by Grant RF57178 from The Rockefeller Foundation.

92

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

was injected to intraperitoneal cavities 30 minutes before X-irradiation.
Samples of ascites fluid were taken 30 minutes after irradiation. As control
the same dose of DNP was injected to non-irradiated mice and samples
were taken one hour after the injection. For the observation of metaphasic chromosomes squash method with water pretreatment (MAKINO,
1959) was adopted, and the number of chromatid and isochromatid breaks
was scored. The result of the experiment is given in Table 1. With the
exception of 100r-irradiation, the breaks were significantly increased by
pretreatment with DNP. The result of this experiment indicates that DNP
inhibits the restitution of the breaks.
Table 1.

Frequency of X-ray induced chromatid breaks by pretreatment
with DNP for 30 minutes. Each of samples was taken
30 minutes after the exposure---

Treatmentwith DNP

Dosage-------_.

Total cells Isochrom. Chromatid
analysed
breaksbreaksTotal
breaksBreaksper cell42370.2±0.05
0.7±0.15-------~--_.-

2
1

40
36

26
33

1
0

68
80

250 r

Non-treatedTreated

50
56

2
1

130
568

132
569

500r

Non-treatedTreated

23
32

0
1

121
290

121
291

Control

Non-treatedTreated-------,.

100 r

__. _

..

198
55

--------._.

_-~-

Non-treatedTreated- - " - - - - - .----

69
80- - - - - - - - - -.. "----2.7±0.66
2.4±0.63._---

2.6±0.48
10.2±1.43--------------_._---- - _ .. , - - - - - - - - -5.3±1.28
9.1±2.21._----

Further experiments were made to investigate the time relation between
breaks and restitutions. In these experiments only 250r irradiation was
employed. The results of the experiments are shown graphically in Figure
1A and 2B. In the irradiated experiment, two kinds of treatments were
given, namely DNP pretreatment 30 minutes before irradiation and DNP
post-treatment immediately after irradiation; no DNP (only X-irradiation)
was applied in the control. Samples were taken immediately, 10 minutes
and 30 minutes after irradiation. In the experiments with DNP pretreatment, a significant increase in the frequency of breaks was observed
immediately after irradiation. Further increases of breaks were observedin the following 10 and 30 minutes. However, no visible change wasfound in the frequency of breaks between irradiated only and the posttreated mice with DNP after 10 and 30 minutes. The results of DNPRESEARCHES CARRIED OUT IN 1960

93

post-treatment indicate that breaks produced by X-rays are restituted ina short time. However, when DNP was applied as pretreatment, restitutions were inhibited and some of the(A)
10
breaks remained open for at least
Irradiated Material (250r)
30 minutes. The remarkable inIrradiated without DNP<:rease in breaks observed 30 minPre-DNP-treatedutes after irradiation indicates thata certain stage, 30 minutes before
metaphase, is more sensitive to
X-ray irradiation than other stages
preceding at least by 10 minutes
the metaphase. On the other hand,the same non-irradiated material
was used for confirmation of theeffect of DNP pre-treatment. The
three kinds of treatments wereapplied in non-irradiated material.
(B)
The frequency of breaks is shown
Non-irradiated Material
Figure lB. From the comparison
a c::J Controlof the results with irradiated andb ~ DNP for 1 hr.
non-irradiated material it follows
c ~. D~-P for 30 min"that the striking number of breaks
Fig. IA. Relation of chromatid breaked
found after 30 minutes in DNP
to time after X-irradiation, with expretreated material is almost enposures made after and before DNPinjection (0.2 ml of IO~2 M). IE. Nontirely due to the inhibition ofirradiatedmaterial treated in similar
restitution of breaks produced byway.
X-rays.

85.

Effect of sodium azide (NaN3) on Xray induced
chromatid breaks ll(By Kazuhiko UTSUMI)

It is known that sodium azide (NaN3) is a respiration inhibitor. As
stated above, DNP, another respiration hinibitor, caused a remarkableincrease in the number of chromatid breaks in Ehrlich tumor cells when
it was injected to tumor bearing animals before X-irradiation.
In order to compare the effect of NaN3 with that of DNP, 0.2 ml of
10- 2 M and 2 x 10- 2 M NaN3 were injected into the intraperitoneal cavityof tumor bearing mice 30 minutes before 250r X-irradiation. The frequencyof breaks per cell was in the control (250r irradiation only) 0.16% and
1)This work was supported by Grant RF57178 from The Rockefeller Foundation.

94

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

2.86% immediately and 30 minutes after irradiation, respectively, and inbatches pretreated with NaN3 it was similar or lower than in the control
batch. It can be concluded that the effect of NaN3 on X-ray induced
chromatid breaks is markedly different from that of DNP.
A possible mechanism of ineffectiveness of NaN3 on chromatid breaks
is discussed in this annual report by Moriwaki, one of our collaborators,
from the view point of ATP content.

86.

Changes of ATP content in Ehrlich tumor cells
after treatment with DNP and NaN3 '1(By KAZDO MORIwAKI)

Measurements of ATP content in cells after treatment with DNP andat the same time observations of chromosomal aberrations in the samematerial have as yet not been carried out. In order to estimate the ATPlevel, the lO/P content of tumor cells was measured following the treatments with DNP and X-rays.
Before X-irradiation no remarkable change was found in lO/P content
in the tumor cells after treatment with 10-2M DNP in vivo. But following irradiation an increase in X-ray induced chromosomal aberrations
could be observed after the same treatment with DNP. In order to estimate accurately changes in ATP content and in 32p incorporation after
DNP treatment, the gradient chromatograph method was employed.
The results indicate an appreciable inhibition of cell metabolism by
DNP, that is, a decrease in ATP content, an increase in ADP and inorganic phosphates (Pi) and an increase in 32p in corporation into ADP
and Pi. No significant changes were found in 32p incorporation into total
nucleic acid and protein.
The reducing effect of DNP on ATP content can be markedly increasedby X-ray irradiation. To avoid individual variation in tumor bearing
mice, one ml from a pool of ascites tumor cells of four mice was used inthis experiment. Probably because of the incubation of tumor cells without addition of nutrients and oxygen, a considerable decrease in 10/P
content was found in the control tube, and more markedly in a tube
treated with DNP. Furthermore, there was beyond doubt an additive effect of X-rays on the lO/P decrease by DNP. A similar effect of X-rays
also appeared in a tube treated with NaN3, although the decline of lO/P
level occurred to a smaller degree.
In view of the above results it is conceivable that in animal cells an1) This':;'work was supported by Grant RF 57178 from the Rockefeller Foundation.

RESEARCHES CARRIED OUT IN 196095

oxidative metabolism with its concurrent formation of ATP is necessary
for rejoining broken ends as previously stated by WOLFF and LUIPPOLD(1955).

87.

Cytological observations of ascites tumors treated withchronic r-radiation ll(By Tosihide H. YOSIDA and Teiichiro TAKAHASHI)

Acquired radioresistance of cancer cells has been discussed clinicallyand experimentally by several authors. Some of them reported a development of radio resistance, whereas others noticed no change in radiosensitivity following irradiations.
The present study was aimed at investigating whether ascites tumor
cells acquire or not radioresistance at chronic irradiation. r-rays werechronically given to the whole body of mice and rats bearing Ehrlich
ascites carcinoma and YOSHIDA sarcoma. A hyperdiploid strain of theEhrlich ascites carcinoma was transplanted into the peritoneal cavity ofmice which were divided into four experimental groups. Each groupconsisted of 3 or 4 animals. One group as a control experiment wasplaced in the usual mouse room and the other groups were chronically
irradiated with 50r, 100r and l50r r-rays per day (22 hours) for 70 days
(10 transplant generations). The total number of mitotic cells gradually
decreased; the number of metaphasic cells was also reduced.
The frequency of anaphasic aberrations, however, increased in the courseof r-irradiations. In order to examine wheter the tumor cells which werepretreated with r-rays have acquired radioresistance, acute irradiation with
100, 300 and Saar X-rays was applied to the total body of tumor bearing
mice. The frequency of cells containing micronuclei, binuclear and polyploid cells, and the mitotic index in pretreted and non-treated tumor cells
were observed from immediately after irradiation to 96 hours later. The
mitotic index: in the non-treated tumor line decreased markedly after 100
to Saar acute X-irradiation, whereas that in pretreated tumor lines decreased
remarkably already after lOOr and 300r X-irradiation, but after saar Xirradiation it was not reduced as strikingly as in the control. The frequency·of anaphasic aberrations in the non-treated tumor line was after acute Xirradiation significantly higher than in the pretreated tumor lines. It isinteresting that the frequency of tetraploid or triploid tumor cells in the
diploid Ehrlich strain decreased by continuous r-radiation. It is assumed
that stem cells have much stronger resistance to r-radiation than othertumor cells.
1) This work was supported by Grant RF57178 from The Rockefeller Foundation.

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ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

YOSHIDA rat sarcoma was chronically irradiated with 2r and 50r per day(22 hours) at a r-field from 6DCO source for 160 days. The chromosome
constitution of tumor cells after this continuous radiation was observed.
52 per cent of tumor cells were characterized by the tumor stem karyotype. Although there were observed various abnormal cells, the stem line
karyotype was not changed by chronic radiation.
Based on the above investigation, it can be concluded that the karyotype
of tumor stem line does not change by chronic radiation, and that thesecells may have stronger resistance to radiation than other non-stemline
cells. Tumor lines which were chronically treated by r-rays showedslightly stronger resistance to acute X-radiation in respect to the occurrenceof abnormal mitosis. Such radiation resistance seems to be due to selection of tumor stem cells which have stronger cell-activity than othernon-stemline cells.

88.

A theory for the frequency distribution of cluster mutants li(By Sohei KONDO)

Many mutants are often found in the offspring of an animal or plant
when irradiation was applied at the early embryonic stage. It is noteworthy that the mutation rate estimated from the observed number ofmutants to a high degree depends on how they are treated.
A general theory for the frequency distribution of the number of mutants
per irradiated individual has been developed on the basis of the following
assumptions: 1) Presence of two types of germ cells with different multiplication factors in each individual when exposed to radiation, 2) Poisson
distribution of the initial mutative events, 3) Cell multiplication without
fluctuation, and 4) Random sampling of gametes giving rise to the offspring. The frequency distribution function of mutants depends on fourparameters which are to be adjusted to fit the experimental data.
The theory has been applied to T AZIMA'S (see T AZIMA'S paper in thisreport) experimental data on silkworm irradiated at their gonial stage.
Comparison of the theoretical distributions with the experimental datashows a very good agreement.
From the theory we have also derived a method for estimating thenumber of the two types of the germ cells; they are of reasonablemagnitudes. As to the estimation of mutation rate, we have reached theconclusion that each mutant of a mutant cluster should be counted as
if it were a single mutant observed per insect.
(Published in Jap. Jour. Genet. 36: 6-17, 1961)
1) This work was supported by Grant RF57178 from The Rockefeller Foundation.

RESEARCHES CARRIED OUT IN 1960

89.

97

A biophysical consideration on radiation mutagenesis' )(By Sohei KONDO)

The sequence of events which leads from the absorption of radiation
to the eventual recovery of mutants, is presented in perspective. The
radiation-initiated mutagenesis has been classified into four steps: (I) Induction of Premutation. (II) Modification of Premutation. (III) Fixationof Mutation and (IV) Detection of Mutation. A model of the mechanismof mutational events in microorganisms, modifiable by post-irradiation
treatments, is proposed and applied to the interpretation of the differencesin mutation rate in various stages of germ cells of the silkworm.
(Published in Progress Theor. Phys. Suppl. No. 17: 129-142, 1961)

F.

RADIATION GENETICS IN PLANTS

90.

Radiation effects of beta- and gamma-raysin Triticum monococcnm 1)(By Seiji MATSUMURA)

Dormant seeds of Triticum monococcum fiavescens were soaked in wateror 32p and 131 1 solutions for 1 and 2 days just before sowing. Radioactive
solutions contained 0.2-1.2 mcjgr of 32p and 0.8-1.6 mcjgr of 1311. To
compare the beta-radiation effects of these solutions with those of gammarays, the seeds were subjected to chronic gamma-radiation from a BOCO
source at 2, 4 and 6 kr during water-soaking. The germination rate,
seedling heigh 4 weeks after sowing, survival rate, mature plant height,
seed fertility and chromosome aberrations in PMC's of the treated plants,
and the chlorophyll mutations in the X 2 were compared. As to the first
5 characters, the effects of beta-radiation from 0.2 mcjgr 32p solution for
2 days corresponded roughly to those of 4 kr gamma-radiation for 2 days,
and were slightly higher than those of 1 day beta-radiation from 0.4 mcjgr
32p solution and of 1 day gamma-radiation at 4 kr.
Also the effects of
1 day beta-radiation from 0.8 mcjgr 32p solution coincided roughly withthose of 6 kr gamma-radiation for 1 day. Further one day beta-radiationfrom 1.6 mcjgr 131 1 solution for 1 day was considerably less effective than
2 kr gamma-radiation for 1 day and showed similar effects as 1 day betaradiation from 0.2 mcjgr 32p solution.
In general, the effects of gamma-radiations on chromosome aberrations- ~ . _ - - _ .

1)----------

This work was supported by Grant RF57178 from The Rockefeller Foundation.

98

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

and gene mutations were unexpectedly small, compared with those ofcorresponding beta-radiations from 32p and 131 1 solutions.
If we assume that the effects of beta-radiation are confined to theembryo, we find by calculation that the 0.4 mc/gr 32p solution for 1 dayor the 0.2 mc/gr 32p for 2 days equals 5 or 4.8 krad, respectively. This,
too, will account for the obtained data.

91.

Segregation ratio and viability of several chlorophyllmutants in einkorn wheat(By Taro FUJII)The segregation ratio often showed a wide range of variation in the
X 2 -generation. The mutant homozygotes appeared very often in too small
numbers, for instance 14:2 in albina-5002, 12:2 in albina-5005 and 19:4 inxantha-5041. Occurrence of more than 25 per cent of recessive homozygotes, as 1: 5 in chlorina-5040, was very rare. But the usual 3: 1 ratio
could be found in the successive generations of 35 of 45 chlorophyll mutant
strains. Among the remaining 10 strains, such as albina-5008, -5012,
striata-5057, basi-viridis-5059, the segregation did not fit the 3: 1 ratio.
Low germination ability of the mutant homozygotes in some strains(striata-5057, basi-viridis-5059) is partly responsible for their deficientnumbers. In all investigated cases the mutation from dominant to recessive concerned single genes. But in some strains, such as basi-viridis-5077,
virido-albina-5072, virido-xantha-5100 etc., albina and striata plants weresegregated from the heterozygotes. A peculiar phenomenon was observedin virido-xantha-5100, namely the occurrence of 76 virido-xanthas, 9 albinas
and 6 striatas besides 59 normal plants. These strains will be further
investigated.
Viability was examined in 65 strains of several chlorophyll mutants,
such as albina, chlorina, basi-viridis, etc. Ability recover the chlorophyll
content was mostly observed in bi-colored mutants, to such as basi-viridis andvirido-albina and did not occur in uniformly colored ones, such as chlorina,
xantha and albina. Neither could the striata mutant recover the chlorophyll content. The recovery occurred only when they were grown in
the greenhouse or in the phytotron and was restricted in the field to thewarm season. In most of the mutants, except chlorina and striata, occurred a more pronounced dilution of green color in the field during thewinter months than at seedling stage and they all died out. But the time
rate of chlorophyll content recovery was different by the strain. Also
1) This work was done under Research Contract No. 27 with the International
Atomic Energy Agency.

99

RESEARCHES CARRIED OUT IN 1960

differences within the same mutant type were observed, for instance inthe recovery speed and viability. May be different steps in the genetic
make-up of chlorophyll production are blocked in the various strains.

92. Preliminary experiments on the relation between dose rate
and radiation effect in einkorn wheafl l(By Taro FUJII)

10 and 20 kr irradiations with three different dose rates were applied toeinkorn wheat seeds. The highest dose rate was 1,000 r/min of X-rays
and the lowest was 0.75 r/min of 10 kr and 1.5 r/min of 20 kr gammaradiation. 33 r/min irradiations at 10 and 20 kr were used with X- as well
as gamma-rays for the check of RBE differences. Then seedling height,
fertility and chlorophyll mutation rate in the X 2 -generation were examined(Table 1).
Table 1.
Dosage(kr)

Effect of radiations with different dose rates

Dose rate(r/min)

o
10

1,000
33 (X-ray)
33 (r-ray)0.75---------

20

1,000
33 (X-ray)
33 (pay)
1.5

Seedlingheight(em)

Fertility in Xl:

11.03

66.54

o

8.52
7.20
8.64
9.03

45.89
44.02
43.94
58.31

8.51
5.96
5.42
4.14

(%)

Chlorophyllmutations in X2(%)- - - - - ---------

1.68
I
1.63
I
3.72
I
I
2.97
_______ L

died
13.07
30.76
31.81

o
8.19
4.55

In all irradiated lots the seedlings were shorter and the fertility on theplants was reduced in comparison with the normal lot, this effect being
milder in the batch of lowest dose rate than in the other three irradiatedlots. 20 kr lot with the highest dose rate was the most severely damaged
and all plants died after germination. The differences were not clear
between intermediate and highest dose rates, especially at 10 kr.
The higher the dose rate at 10 kr, the higher was the chlorophyll mu-----------

1) This work was done under Research Contract No. 27 with the International
Atomic Energy Agency

100

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

tation rate in the X2-generation. This tendency was also observed in
20 kr gamma-irradiations. That no mutation was found at the intermediate
X-ray dose rate will be due to the small number of X 2 head progenies.
Similar mutation rates were obtained at 10 kr from the intermediate dose
rate in X- and gamma-rays. RBE of X- and gamma-rays was about thesame as for mutation rates.

93.

Relation between radiation effects and dose rates in rice
(By

Tomoo MABUCHI, Taro FUJII and Seiji MATSUMURA)

In order to investigate the relation of radiation effects to dose rates,
dry seeds of upland rice were irradiated by gamma-rays from a 60CO or
137CS source at the doses of 5, 10, 20 and 30 kr and three different dose
rates of 10 r/hr, 300 r/hr and 10,000 r/hr. Only the 10 rfhr irradiation at
30 kr was not applied. These chronic, moderate and acute irradiations
were terminated almost simultaneously before sowing. The germination
rate, seedling height 20 days after sowing, seed fertility and contents offree amino acids and reduced sugar were investigated. At 20 and 30 kr
the germination rate was slightly lower, especially at chronic 20 kr andacute 30 kr irradiations, than in the untreated lot and those with 5 and
10 kr irradiations. Unexpectedly, chronic irradiation, which was applied
from the start, was most effective in inhibiting the growth of seedlings
and in decreasing the fertility, especially at 20 kr. Thus an intensificationof radiation damage due to storage was found.
Contents of free amino acids and reduced sugar were examined in 5
grams of fresh leaves 20 days after sowing. There was no marked difference between the content of the first and the second leaf. Neither
the contents were noticeably influenced by the dosage. But at the same
dosage chronic irradiations showed higher contents of amino acids than
the acute ones, while the contents of reduced sugar were not clearly
different by the dose rates. The relation between growth of seedlingsand dose rates was reversed to that between amino acid contents anddose rates. From these findings it is suggested that the protein synthesis
from amino acids is depressed at low dose rates, while high dose rates
inhibit the amino acid metabolism from carbohydrates through organic
acids, i. e. the preceding steps of protein synthesis.

RESEARCHES CARRIED OUT IN 1960

94.

101

Effects of radiation on the susceptibility of wheat
and rice seedlings to leaf rust
(By Keizo KATSUYA)In Triticum Spelta, T. Timopheevi, T. vulgare and Oryza sativa a batchof seedlings with 2 leaves was irradiated by X-rays and gamma-rays at
10, 30, 50 and 70 kr. The seedlings were inoculated 1 and 7 days after
irradiation by spraying with an aqueous uredospore suspension (Puccinia
triticina 21B) and the effects of radiation on the susceptibility to leaf rust
were observed. In the remaining seedlings of the same age the first andsecond leaves were cut off and the amounts of amino-nitrogen and reduced
sugar were measured.
Effects of irradiation on leaf characters, namely stomata size, internal
structure and viability, and sporulation capacity on irradiated leaves were
examined. Rust susceptibility of irradiated plants (T. Spelta and T.
vulgare) was increased, as compared with the normals. In T. Timopheevithat was inoculated 7 days after irradiation, the third leaf was susceptible,
while the first and second leaves of the same plants were mostly resistant.
Rust susceptibility of irradiated rice plants remained unchanged.
In T. Timopheevi seedlings, the amount of amino-nitrogen increased in
the seedlings 1 day after irradiation, as compared with that of the normals, while it decreased 7 days after irradiation. The amount of reduced
sugar increased in seedlings 1 and 7 days after irradiation in comparison
with the normals. Stomata size and internal structure of irradiated plants(T. Spelta and T. vulgare) did not change noticeably, as compared with
the normals. Effects of irradiation on the viability of leaves were tested;when the growing point suffered radiation damage, the already developed
leaves remained longer fresh than those of the normals. The uredosoriof leaf rust on irradiated T. vulgare were larger than on normal plants.
Relationships between rust susceptibility and radiation await further experiments.

95.

Calculation of absorbed dose delivered to wheat seeds
soaked in 32p and 131 1 aqueous solutions l1(By S. KONDO and H. ISHIWA)

We have already confirmed that the coats of wheat seeds soaked in 32p
or 131 1 aqueous solutions prevent strongly radioactive isotopes from penetrating into the interior of the seeds at least for one to two daysll.
I) This work was done under Contract No. 27 with the International Atomic
Energy Agency.

102 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

Therefore the dosimetry of seeds soaked for one or two days can bemade with a good accuracy by taking account of the contribution of theisotopes assumed to be homogeneously distributed outside the seeds.
Roughly speaking, the seeds of Triticum monococcum flavescens soaked inwater for 24 hrs have an ellipsoidal shape with the three axes 2.8, 3.8,
7.9 mm long. In this report, however, we will calculate the average
embryo dose and the average seed dose based on simplified models.
[1] Average Embryo Dose: Assume that the dose to the embryo ofthickness h (g/cm 2 ) can be approximated by that to a surface layer ofthickness h of an infinite plane slab made of a tissue-like material with
infinite thickness whose plane surface is in contact with an infinite aqueous solution with homogeneously distributed p-emitting isotopes. Then,integrating Loevinger's empirical formula 2 ), the average dose D(fi5 isgiven by.... - = -1 D{3 -a {c 2[ 3h--(e-el-lc5 -I)h 2 - 1 I)hI2 n -C-]Vh / C1 ) - D(h)
2
h
I)
4 c
2 c
I)h

+ ~ (e-e ->h) } ;1

O<h::; ~=J..D{3~{£(ll-e)+J..(e-el->h)};
2

h

I)

4

I)

(1)

..£<h
I)

where I) (cm 2/g) is the apparent absorption coefficient of the p rays, a andc are the characteristic constants of the p-emitting isotopes and the D{3is the absorbed dose in the interior of the isotope solution. The D{3 isgiven byD{3=51.1E{3aoC-;-At) rad(2)

where E{3 (Mev) is the average p-ray energy, A (day-I) the disintegration
constant, ao (p.c/g) the initial concentration of the p-active isotopes in thesolution and t (days) the soaking time.
Since, for 32p, a=1/3, c=l, 1)=9.2 cm 2/g, E{3=0.694 Mev and A=0.693/14.3
day-I; and for 131 1, a=0.26, 1)=40 cm 2/g, E{3=0.187 Mev and A=0.693/8.0
day-I, from equations (1) and (2) we have for h=0.063 g/cm 2 , the average
thickness of the embryo soaked for one day:
32P:

15embryo=0.355D{3=13.1 kr/day/mc/ml=25. 6kr/2 days/mc/ml

(3)

1:

Dem bryo=0.143D{3=1.39 kr/day/mc/ml=2.66 kr/2 days/mc/ml

(4)

131

where we have used the value of 0.945 rad/r for Co-60 r rays calculated
for the elemental composition 4 ) of the seeds.

103

RESEARCHES CARRIED OUT IN 1960

[2] Average Seed Dose: Assume the average dose to the whole seedmass can be approximated by that to a sphere with radius b. Then,
using the published numerical calculations 2 ), we can easily calculate theaverage seed dose Dsph(5).

If we assume b=0.22 (g/cm 2 ), we have

32P:

Dseed =0. 54Dfl =19. 2 kr/day/mc/ml=37.5 kr/2 days/mc/ml

(5)

131

Dseed =0.14D fl =1.36kr/day/mc/ml=2.6 o kr/2 days/mc/ml

(6)

1:

[ 3] Comparison with Experimental Data: Comparing with MATSUMURA'S3)
data for soaking the seeds in 32p and 131 1 aqueous solutions for two days
and two day exposure of soaked seeds to 60CO r rays, we obtain thefollowing table.
Considering the partial shielding of (3 rays by the proximity of seeds
Table 1.

Comparison of the data of r-irradiated 32p or 1311 /'i-rayed
experiments with the theoretical estimations-

Experimental equivalent
mc/ml ___~oentgen_(krL
Seedling heights
Fertility

Isotope
32p
32 P
131 I

i

0.15
0.30
0.6

2.0
4.8
1.6
----

3.7
4.4
1.9

-

--

Calculated equivalent
roentgensJkr)
Embryo dose

Seed dose

3.9
7.8
1.6 (1.9)*

5.6
11.3
1.6 (1.8)!

-* The figures in the parentheses represent the total exposure doses corrected
for r rays of 1311.

touching each other when they are soaked in the isotope solution inbatches of 20 seeds per a gauze pouch, from Table 1 we may conclude
that the present calculation gives a fairly good estimation of the absorbed
in the soaked seeds, because the RBE values of these (3 rays relative to
Co-60 r rays will be close to unity.
1)
2)

S. Kondo et al. Ann. Report, National Inst. Genetics (Japan) No.8, 98 (1958).
R. Loevinger et al. in Radiation Dosimetry (G. ]. Hine and G. L. Brownell, ed.)Academic Press. 1956, p. 694.
3) S. Matsumura. Ann. Report, National Inst. Genetics (Japan) No. 10, 136 (1960).
4) S. Kondo. Ann. Report, Nationl Inst. Genetics (Japan) in Japanese, No.9, 134(1959).

104

ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11

96.

The effect of X-irradiated pollen grains on the formationof empty hybrid seed in nicotiana(By Masao TANAKA)

In the cross, Nicotiana tabacum (if) x N. alata (~) in which only empty
seeds are expected, X-irradiated pollen grains increase the percentage offilled seeds markedly. The author had found in the ovaries of tobacco
dusted with pollen grains of N. alata, that auxin levels were very lowalmost at the same level as those of the ovaries kept without pollinationand that in the ovaries dusted with X-irradiated pollen grains, the levelsof auxin rose a little higher.
In order to reveal the action of growth regulators on the fullness ofseeds, the levels of auxins and IAA-inhibitors in the ovaries were investigated. When the partly purified extract from immature capsules is developed on chromatograms using isobutanol-metanol-water (80:5:15) as adeveloping solvent, two spots of auxins usually appear in the position of
Rf 0.19 and 0.97 after spraying with Salkowski-Link's reagent. Twoyellowish white spots of IAA-inhibitors appear in the position of Rf 0.20
and 0.28 on the red background of the paper strips when the original
extract has been developed by the same solvent and treated with SalkowskiLink's reagent after spraying with a diluted solution of IAA. The IAAinhibitors are supposed to be ferment-like substances from the fact thatthey are fluorescent in ultra-violet light and inactivated by KeN andfurthermore, they give purple colour reaction by spraying with a solutionof nynhydrin.
The areas of the spots were compared among the extracts of the capsules, N. tabacum selfed, N. trtbacum dusted with non-irradiated as wellas irradiated pollen grains of N. alata. The spots of auxins from thecapsules dusted with irradiated pollen grains were obviously larger thanthose from non-irradiated pollen grains though they were much smaller
than those from the capsules of selfed N. tabacum. No differences wereobserved in the areas of the spots of IAA-inhibitors among the capsulesof selfed N. tabacum and those obtained from dusting with non-irradiatedpollen grains of N. alata. But the spots from the capsules obtained by
dusting with irradiated pollen grains were obviously smaller than thosefrom the other two kinds of pollination.
Summarizing the results mentioned above, we may draw the conclusionthat the empty hybrid seeds arise partly by a decrease of auxin level
which might be caused by a physiological disharmony between paternal
and maternal genomes in the endosperm. Irradiated pollen grains aresupposed to overcome the decrease of auxin levels by suppressing the

RESEARCHES CARRIED OUT IN 1960

105

action of an inhibitor producing systems and will work toward increasing
the filled condition of seeds.

G.
97.

TECHNICAL NOTE

A preliminary calculation of depth dose curveof 60CO r-ray irradiation(By S. KONDO and H. ISHIWA)

It is well known that the absorbed dose varies appreciably with depth
in the surface region of materials exposed to high energy photons. A
theoretical calculation of the depth dose curve for Co-60 r irradiation oftissue-like materials has been carried out.
Assume a thin block of material with an infinite plane surface. Take
the origin of x coordinates at the surface with the positive x axis parallelto the inward normal and expose the block to r rays coming parallel tothe x axis from the negative side. Then, the absorbed dose dD/dx atthe depth x (g/cm 2 ) due to recoil electrons originating from the surface
and within the solid angle element dQ in the direction of the angle ofelectron recoil, 0, is given by

ED =c1E(()p(_X_, E)--~dQ
dx
J
cos 8
R(E) cos 8(1)where c is the proportionality constant, E(8) the energy of recoil electrons,
R(E) the maximum range of electrons with energy E, f(8)dQ the numberof the above recoil electrons and P(x/cos 0, E) the fraction of energy released by the above electrons per unit thickness at distance x/cos 8 fromthe surface along the 8 direction. The relative depth dose P(f;, E) at thedepth f;( =x/cos 8) for electrons with incident energy E and angle 8 =0,
has been calculated by SPENCER'). However, to simplify further the calculation, we will use as a preliminary method NAKAI'S approximation 21 whichfits well SPENCER'S theory and the experimental results for aluminum:P(f;, E)=exp [-4.532(f;-0.336R)2/R2](2)

where, for simplicity, the extrapolated range R used by NAKAI 21 has been
replaced by the maximum range R.
Now, the relative depth dose !(x) at depth x is given by

I(x) = IX dD(x) dx/ loo dD(x) dx
Jo dx
Jo dx

(2 )

106 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11
The depth dose curve calculated from equation (3) is given by Curve III
in Fig. 1. Using the experimental spectrum of secondary electrons ob·
tained for aluminum at 8=0 and 15 by AGLINTSEV et at. 4 ), we haveobtained the relative depth dose curve IV (see Fig. 1) for the external
secondary electrons originating from the materials outside the dosimeter.
The final curve I is the sum of curves III and IV where curve IV has
been adjusted to fit the experimental curve II obtained by ONAI et at. 3 )15
at x=0.2. The agreement between\
the theoretical and experimental\'"
curves is satisfactory. An improve""'_':.__
ment of the present calculation by-------_
using SPENSER'S theory is now under10.",--;......
way.
~./
An example of a very high conI:?
'r~ ,/tamination by external secondary~ 0.:, r im/ /electrons is given in curve V forI
the case of the 60CO r irradiation//....
facility with no filter in NationalIV ••• , . . . . . . . . . . . . . . . . . . . •Institute of Genetics. Irradiation of/•small animals or plant seeds should
05
U.2
0·3
04
01
be carried out by encasing the mao
Depth (glcm
terials in a container with electronic
equilibrium wall thickness not only for the secondary electron equilibrium
but also for elimination of the external secondary electrons.
0

0

2)

1)
2)
3)
4)

L. V. SPENCER:

N. B. S. (U. S. A.) Monograph 1 (1959).
Y. NAKAI: unpublished.
Y. ONAI et al.: Rinsho Hoshasen 3, No.9 (1958) 56.
K. K. AGLINTSEV et al.: Atomnaia Energia 2, Feb. (1957) 66.

BOOKS AND PAPERS WRITTEN BY STAFF MEMBERS

107

BOOKS AND PAPERS PUBLISHED IN 1960
BY STAFF MEMBERS
CHANDRARATNA, M. F. and K. SAKAI 1960 A biometrical analysis of matrocinous
inheritance of grain weight in rice. Heredity 14 Parts 3 & 4, 365-373
DOIDA, Y. 1960 Developmental studies in the genus Polygonum I. Microsporogenesis of Polygonum persicaria L. Bot. Mag. 73: 278-282
1960 Cytological studies in Polygonum and related genera I. Bot. Mag.
73: 337-340
ENDO, T. 1960 Biochemical genetics in higher plants (in Japanese). Protein,
Nucleic acid and Enzyme 5: 75-86
1960 Paperchromatography and its application to the plant science in"Experimental Methods in Cytogenetics" (ed. by I. NISHIYAMA Yoken-d6) 381434
FUJII, T. 1960 Radiosensitivity in plants IV. Experiments with several mutant
strains of einkorn wheat. Jap. Jour. Genet. 35: 110-119
1960 Mutations in einkorn wheat induced by X-rays VI. Segregation
ratio and viability of several chlorophyll mutants. Seiken Zih6 11: 12-20
and Y. ONO 1960 Amino acid content of einkorn wheat chlorophyllmutant (Preliminary report). Proc. Jap. Acad. 36: 612-617
FURUSATO, K. and A. MIYAZAWA, 1960 Breeding of amphidiploid by cross between Nicotiana rustica and N. tabacum (in Japanese). Hatabako Kenkyu 25:
82-85
1959 A new method to produce easily rooting cuttings (in Japanese).
Seiken Zih6 10: 95-97
1960 Investigation of agriculture in Africa (in Japanese). Kankitsu 11:
46-50, 12: 108-111; 60-64; 56-61
1960 Investigation of Citrus in Africa (in Japanese). Kajitsu Nihon
15: 4-5; 53-55
1960 An example of seedless fruits by gibberellin treatment (in Japanese).
Iden 14: 61
HIRAIZUMI, Y. and JAMES F. CROW 1960 Heterozygous effects on viability, fertility, rate of development, and longevity of Drosophila chromosomes that arelethal when homozygous. Genetics 45: 1071-1083
L. SANDLER, and JAMES F. CROW 1960 Meiotic drive in natural populations of Drosophila melanogaster III. Populational implications of the Segregation-Distorter locus. Evolution 14: 433-444
and - - - - 1960 Meiotic drive in natural populations of Drosophilamelanogaster IV. Instability at the Segregation-Distorter locus. Genetics 45:
1269-1287, I. FUKUDA, T. NARISE and M. KURABAYASHI 1960 Evolution and Variation in Trillium VI. Migrations among natural populations of T. kamtschaticum
across the Ishikari Depression. Evolution 14: 224-231
KAO, K. N., C. H. Hu, W. T. CHANG and H. I. OKA 1960 A biometrical-genetic
study of irradiated populations in rice: Genetic variances due to different

108 ANNUAL REPORT OF NATIONAL INSTITUTE OF GENETICS NO. 11
doses of X·rays. Bot. Bull. Acad. Sinica 1: 101-108
KATAYAMA, T. 1960 Studies on the intercellular spaces in rice I. Proc. Crop
Sci. Soc. Jap. 29: 229-233
KATSUYA, K. 1960 Susceptibility of the albina mutant of Einkorn wheat to leaf
and stem rusts, Puccinia triticina and P. graminis. W. I. S. No. 11: 16
KAWAHARA, T. and S. ICHIKAWA 1960 Studies on heterosis in the domestic fowl
II. A comparative analysis of body weight in F[ hybrids and their purebreds(in Japanese with Eng. resume). Jap. Jour. Zootech. Sci. 31: 109-114
1960 Studies on heterosis in the domestic fowl III. Mortality and susceptibility to leucosis in F 1 hybrids (in Japanese with Eng. resume). Ibid. 31:
115-118
KIHARA, H. 1960 Principles of genetics I (in Japanese). Iden 14: 54-56
1960 Principles of genetics II (in Japanese). Iden 14: 52-55
1960 The rice plant in Sikkim. A report of the scientific mission. Seiken Ziho 11: 46-54
Y. OHTA and N. YUTANI 1960 Comparison of hereditary variegation
and breaking by viruses (in Japanese). Jap. Jour. Gen. 35: 274
and K. TSUNEWAKI 1960 Production of polyploid wheat by nitrous
oxide. Proc. Jap. Acad. 36: 658-663
KIMURA, M. 1960 Genetic load of a population and its significance in evolution(in Japanese with English summary). Jap. Jour. Genet. 35: 7-33
1960 Optimum mutation rate and degree of dominance as determined by
the principle of minimum genetic load. Jour. Genet. 57: 21-34
1960 Outline of population genetics (in Japanese). Baifu-kan- -- 1960 Relative applicability of the classical and the balance hypotheses toman. Especially with respect to quantitative characters. Jour. Radiation
Res. 1-2: 155-164.
KONDO, S. 1960 Radiation Dosimetry for Biology and Chemistry (in Japanese).
Biophys. Biochem. Symp. 5: 229-245 Nanko-do- ---- - 1960 Neutron response of silver-activated phosphate glass. Health Phys.
4: 21-24----- and M. L. RANDOLPH 1960 Effect of finite size of ionization chambers
on measurements of small photon sources. Rad. Res. 13: 37-60~----- 1960 Absolute dosimetry with silver-activated phosphate glass for liquidand solid containing fJ-ray sources (in Japanese). Proc. 3rd Jap. Isotope Congress465-469~~- 1960 Dosimetry for radiation chemistry (in Japanese).
Ko-bunsi 2:
3-14~~-- 1960 Principles or radiation dosimetry (in Japanese). Gensiryoku Kogyo
6: 74-80
MATSUMURA, S. 1960 Radiation genetics in wheat V. Influence of irrradiation time
and temperature on the genetic effects of ionizing radiation in diploid wheat.
Jap. Jour. Genet. 35: 197-204.
1960 Radiation effects of fast and thermal neutrons on wheats I. Genetic
effects of neutrons on einkorn wheat. W. I. S. No. 11: 5-7
1960 Comparison of radiation effects of beta- and gamma-rays on einkorn

BOOKS AND PAPERS WRITTEN BY STAFF MEMBERS

109

wheat. W. I. S. No. 11: 12-13
and T. FUJII 1960 Induction of bud sports of radiation (in Japanese).
Proc. 3rd Conf. Radioisotopes 1959: 1005-1008
and S. KONDO 1960 CoGO gamma-irradiator and its use inplant genetics. Large Radiation Source in Industry, IAEA 2: 179-193
MIKAMI J. and Y. OGAWA 1960 Effects of Glucuronic acid on the function of liver
after partial hepatectomy (in Japanese). Advances in Glucuronic acid Res. 19551959, 123-126
MORISHIMA, H. and H. I. OKA 1960 The pattern of interspecific variation in thegenus Oryza: Its quantitative representation by statistical methods. Evolution
14: 153-165
MORIWAKI, K. 1960 Effects of the content of energy-rich phosphate esters on theswelling in mouse diaphragm. Ann. Zool. Jap. 33: 211-216
MUKAI, T. 1960 Destiny of heterotic lethal genes in populations (in Japanese).
Jap. Jour. Genet. 35: 278-279
1960 Population dynamics of selfing. Seiken Zih6 11: 43-45
NAKAI, S. 1960 Experimental methods in research of radiation genetics. "Experimental Methods in Cytogenetics" (ed. by I. NISHIMURA) Yokendo 435-533
NAWA, S. 1960 The structure of the yellow pigment from Drosophila. Bull. Chem.
Soc. Japan 33: 1555-1560--- 1960 Biochemical genetics of insects (in Japanese). Protein Nucleic Acid
Enzyme 5: 87-94
NEzu, M. 1960 Radiation effects of fast and thermal neutrons on wheat II. Relation of ploidy to chromosome aberrations. W. I. S. No. 11: 7-8- , T. KATAYAMA and H. KIHARA 1960 Genetic study of the genus Oryza
I. Cross ability and chromosomal affinity among 17 species. Seiken Zih6 11:
1-11
OGAWA, Y. 1960 Growth, differentiation and regeneration XVI. Synthesis of myosinin the regenerating limb of Triturus pyrrhogaster (in Japanese). Med. and
BioI. 54: 189-192
1960 Growth, differentiation and regeneration XVII. Effect of X-irradiation on the synthesis of actin in early embryo of Triturus pyrrhogaster, Boie(in Japanese). Med. and BioI. 55: 34-36
1960 Synthesis of contractile proteins in the X-irradiated embryo. Nature
186: 77-78- - - 1960 Individual differences of taste-sensitivity to citbittol A, a bitter
substance extracted from the fruit of Colocynthe (in Japanese). Med. and BioI.
55: 91-94--- 1960 Effect of Glucuronic acid on the growth of embryo and the regenerating limb tissue of Triturus (in Japanese). Advances in Glucuronic acid Res.
1955-1959, 25-31-- - 1960 Growth, differentiation and regeneration XVIII. Growth promoting
effect of Na-glucuronate on the early embryos of Triturus pyrrhogaster, Boie(in Japanese). Med. and BioI. 55: 161-164- - - - 1960 Growth, differentiation and regeneration XIX. Synthesis of skeletal
muscle protein in the regenerating tissues of fore limb and tail of Triturus

110

BOOKS AND PAPERS WRITTEN BY STAFF MEMBERS

pyrrhogaster, Boie (in Japanese).

Med. and BioI. 56: 37-39
1960 Taste-ability for phenyl-thio-carbamide of Japanese (in Japanese).
Med. and BioI. 56: 156-159
1960 Growth, differentiation and regeneration XX. Effect of Natrium
glucuronate on tre impediment of growth of X-irradiated embryos of Tritunts
pyrrhogaster, Boie (in Japanese). Med. and BioI. 57: 23-26
1960 Growth, differentiation and regeneration XXI. Effect of X-irradiationon the synthesis of myosin in skeletal muscle of early embryo of Trituruspyrrhogaster (in Japanese). Med. and BioI. 57: 133-136
1960 Effects of gluculonic-acid and related compounds on the growth ofthe embryo of Triturus pyrrhogaster (in Japanese). Med. 17: 894-899
OHTA, T. and S. MATSUMURA 1960 Cytological and histological observations on
the male sterility in sugar beet (in Japanes with Eng. resume). Seiken ZiM 11:
77-81
aNa, S. and S. KONDO 1960 Molecular theory of surface tenion in liquids. Handbuch der Physik )Springer-Verlag/Berlin) 134-280
aKA, H. I. 1960 Variation in competitive ability among rice varieties (Phylogenetic
Differentiation in Cultivated Ric. 19). Jap. Jour. Breed. 10: 61-68
and W. T. CHANG 1960 Survey of variations in photoperiodic response
in wild Oryza species. Bot. Bull. Acad. Sinica 1: 1-14
OSHIMA, C. and O. KITAGAWA 1960 Frequencies of second chromosomes, having
deleterious effects on viability when homozygous in natural population of D.
melanogaster. D. I. S. 34
and1960 The pre-adult viability of heterozygotes of D. melanogaster second chromosomes that are lethal and semi-lethal when homozygous.
D. I. S. 34
SAKAI, K. I. 1959 Practical purity and the method of maintenance of establishedvarieties of rice. News Letter, International Rice Commission 8: 7-13
1960 Studies on the method of selection in the coconut palm. Bull.
Fac. Agr., Tamagawa Univ. 63-72
SHIMOTUMA, M. and Y. OGAWA 1960 Cytogenetical studies in the genus citrullus
III. Bitter substances in fruits of C. Colocynthis Schrad. and C. vulgaris Schrad.
Jap. J. Genet. 35: 143-152
TAIRA, T. 1960 A biochemical study on allelism at Henna locus in Drosophilamelanogaster. Jap. Jour. Genet. 35: 344-350
1960 Is the Hn r3 ry, double recessive mutant a synthesized lethal strain.
D. I. S. 34:
TAKENAKA, Y. 1960 The northern limit of distribution of tropical and subtropical
species in Japan. BioI. Jour. of Nara Women's Univ. 10: 36-39
TAZIMA, Y. 1960 Application of radiations for animal breeding (in Japanese). Kagaku 30: 22-26
1960 Comparison of radiation-induced mutation rates between acute andchronic irradiation in silkworm. United Nations Document AjAc, 82jG/L, 473
TSUJITA, M. 1960 Alkaloids in tobacco plant and sericulture. Kinugasa Gakuho
8: 3-5TSUNEWAKI, K. 1960 Monosomic and conventional gene analysis in common wheat

BOOKS AND PAPERS WRITTEN BY STAFF MEMBERS

III

Lethality. Jap. Jour. Gen. 35: 71-75
and E. G. HEYNE 1960 The transmission of the monosomic conditionin wheat var. Chiness Spring. Jour. Hered. 51: 63-68
and B. C. JENKINS 1960 A comparative study of various methods ofroot-tip preparation in screening wheat aneuploids. Cytologia
YAMADA, Y. 1960 Observations of genotype-environment interaction in production
traits in chickens. Jap. Jour. of Breeding 10: 23-28
1960 Optimum stracture of poultry breeding flock which maximizing
genetic improvement by selection. Jap. Joor. of Genet. 35: 187-196
YOSIDA, T. H., T. ISHIHARA and T. HAMADA 1960 Chromosomal alteration andthe development of tumors III. Change in ploidy of the stemline chromosomeswith a transplantability shift. Gann 51: 55-65
and T. ODASHIRO 1960 Ibid IV. Comparative idiogram analysis
in cells of normal rat liver and rat ascites hepatoma No. 7974. Jap. Jour.
Genet. 35: 35-40
1960 Ibid V. Temporary proliferation of tetraploid cells in the Yoshidasarcoma and their origin. Gann 51: 359-369
and S. TSUJI 1960 Ibid VI. Difference in invasiveness of the diploand tetraploid cells of the Yoshida sarcoma. Jap. Jour. Genet. 35: 377-381--, T. TABATA and H. HIRUMI 1960 Cytological studies on radiation effects
1. Chromosome breaks in the three types of chromosomes of Yoshida sarcoma
cells and their localization. Jour. Rad. Res. 1: 1-9
and H. HIRUMI 1960 Cytological study on the effects of 8-azaguanine
and related compounds on the Yoshida sarcoma cells. Gann 51: 345-357, T. TABATA, T. TAKAHASHI and H. HIRUMI 1960 Effect of X-rays
on cell muclei of ascites tumors, with special emphasis on the chromosome
breaks and reunion (in Japanese with Eng. resume) Symp. Cell. Chern. 10:
203-220and T. ODASHIRO 1960 The walker rat carcinoma having a triploid
stemline (in Japanese with Eng. resume). La Kromosome 44-45
III.

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ACKNOWLEGMENTS
The editor wishes to acknowledge Dr. F.A. LILIENFELD for reading the manuscripts(Yoshito OGAWA).

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