NATIONAL INSTITUTE OF GENETICS
JAPAN
.J

ANNUAL RI:PORT
No. 101959

Published by

THE NATIONAL INSTITUTE OF GENETICS
Misima, Sizuoka-ken, Japan1960

Annual Reportof theNational Institute of Genetics
No.10,

1959

Published by

The National Institute of Genetics, Japan
1960

CONTENTS
General Statement
Abstracts of diary for 1959..........................................
Staff
,
,
, .. .. .. .. .. .. .. .. .. .. .. ..
Council," ..
Research program for 1959 .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Joint researches supported by the grant from the Rockefeller Foundation
Foreign visitors in 1959..............................................
Researches carried out in 1959

Genetics, biochemistry and cytology of insects.
Studies on the E and U allelic genes in the silkworm, withspecial reference to the attachment form between the VIthand XIVth chromosome (TSUJITA)..........................
2. Genetic and biochemical studies on the new white-egg mutant
in the silkworm (TSUJITA).. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..
3. X-ray induced Nl-type mutants of the silkworm (TSUJITA) .. ..
4. Genetic maps of the eighth linkage group of the silkworm(TAZIMA, OZAWA, INAGAKI and KOBAYASHI)
5. Structure and function of pleiotropic genes of the silkworm III.
Genetical study of four mutant strains induced independently
from the Efo}l gene. (TAJIMA and INAGAKI).. .. .. .. .. .. .. .. ..
6. Genetic variations in DDT-resistance of laboratory strains andnatural populations of Drosophila melanogaster (OSHIMA).. . .
7. Frequencies of deleterious chromosomes in natural populationsof Drosophila melanogaster (OSHIMA and KITAGAWA)
8. Effects of X-irradiation on biosynthesis of nucleic acids in
Drosophila (TAIRA and MORITA) .. .. .. .. .. .. .. .. .. .. .. .. .. ..
9. Distribution of the "Sex-Ratio" agent in the tissues of adultsof Drosophila willistoni (SAKAGUCHI and POULSON)
10. Mass migrating activity in inbred lines derived from four wild
populations of Dro:,ophila melanogaster. (NARISE, SAKAI and
IYAMA)..
11. Genetical aspects of two colour mutants of rats caught in thesuburbs of Misima (YOSIDA).. . . . . . . . . . . . . . . .. . . . . .. . . . . .• . .
12. Study on the new mutant" falter" found in the house mouse(Y OSIDA)

1
1
2
4
4
6
9
10

A.

1.

10
13
16
17

18
21
23
25
26

28
29
30

CONTENTS

ii

13.
14.
15.
16.

Peripheral blood analyses of various strains of mice and rats
(HAMADA, T ABATA, KURITA and YOSIDA)
Reciprocal transplantation of skin between rhino and non-rhino
mice (TSUZI and YOSIDA)
Study on phosphatase activity in rhino mouse skin (MORIWAKI)
Effect of energy-rich phosphate esters on the swelling of mouse
diaphragm (MORIWAKI)

B.

Genetics, cytology and taxonomy of ceral crops and related plantgroups17.

Interaction of Aegilops cytoplasm and various wheat nuclear
complements (KIHARA).. .. .. . . . . .. . . .. . . . . . . .. .. .. . . .. .. .. ..
Genome analysis in the genus Oryza (KIHARA and NEZU).. .. ..
Application of SUMP method in taxonomic studies in Oryza(KIHARA and KATAYAMA).. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..
Studies on germination in Oryza (KATAYAMA).. .. .. .. .. .. .. .. ..
Studies on the flowering time of genus Oryza (KATAyAMA)....
Photoperiodic responses of Oryza species (II) (KATAyAMA)......
Monosomic analysis of lethal factors in common wheat (TsuNEWAKI)
Developmental studies in the genus Oryza I. The process ofembryo sac formation (DOIDA).. .. . . . . . . . . .. . . .. . . .. . . .. .. ..
The karyotype and intra-genome pairing of chromosomes in
haploid plants of Oryza glaberrima (Hu and TAKENAKA) ....
Systematic studies in the genus Oryza by statistical methods(MORISHIMA and OKA).. .. .. .. .. .. .. .. .. .. .. .. .. .. ..
Variation studies in wild rice species, Oryza perennis and O.
sativa f. spontanea, by multivariate analysis (MORISHIMA and
OKA)
.. ..
..
Hybrid sterility relations among cultivated and wild strains ofrice (HINATA and OKA).
Variation in hybrid sterility with certain test strains within
populations of wild rice (OKA and CHANG), .. .. ..
An investigation of floating habit in wild rice species (MORISHIMA, HINATA and OKA).. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..
Variation in seed dormancy of wild rice species (MORISHIMA
and OKA)............................
..
Variation in photoperiodic response among wild rice populationsof Thailand (OKA and CHANG) .. .. .. .. .. .. .. .. .. .. .. .. .. ....
The impact of cultivation on populations of wild rice, Oryza
sativa f. spontanea (OKA and CHANG)
Distribution of the glutinous gene in wild and cultivated

18.
19.
20.
21.
22.
23.
24.
25.
26.
27.

28.
29.
30.
31.
32.
33.
34.

31
32
33
34

36
37
39
40
42
43
45
46
47
48

49
51
53
54
55
56
57

_

CONTENTS

35.
36.

.~

37.
38.
39.
40.
41.
42.
43.
44.
45.
46.

47.
48.
49.
50.

populations of rice in Thailand (OKA) .. . . . . . . . . . . . . . . . . . . . .
Population-genetic studies of wild rice of Taiwan (OKA andCHANG)
Variation in yielding capacity among isogenic lines of rice
(OKA).. .. .. .. .. .. .. .. .. .... .. .. .. .. .. .. .. .. .. .. .. .. .. ..
Genetic variability in established varieties of rice (SAKAI)
Variability of seed size characters in wild rice (SAKAI, NARISE
and IYAMA) .... ...... ........ .. .... .. ...... .. .. .. .... ......
Variation in anthesis in wild rice populations (Oryza sativa f.
spontanea) collected in India and Ceylon (NARISE and lYAMA)..
Further note on natural crossing in wild rice (SAKAI and
NARISE).. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..
Competition experiment with wild rice (NARISE and SAKAI).. ..
Propagating capacity and competitive ability of the F, hybrid
between upland rice and" red rice" (IYAMA)
Seasonal sterility of rice in Ceylon (SAKAI).. .. . . .. .. . . .. .. . . ..
Studies on a laboratory method of testing salinity resistancein rice varieties (SAKAI and RODRIGO)
Variation in susceptibility to Piricularia disease in wild rice
populations (NARISE)
Susceptibility of nullisomic wheat dwarfs and their respective
gigas-plants to leaf rust, Puccinia triticina (MATSUMURA and
KATSUYA) .. .. .. .. .. .. .. .. .. . . .. . . .. .. .. . . . . . . .. .. . . .. .. . . ..
Recovery of chlorophyll content in some mutants in einkorn
wheat, Triticum monococcum fiavescens (FUJII)
Amount of amino acids in the chlorophyll mutants of einkorn
wheat (ONO and FUJII)
Susceptibility of wild rice to blast fungus, Piricularia Oryzae(KATSUYA)
Susceptibility of the albina mutant of einkorn wheat to leaf andstem rusts, Puccinia triticina and P. graminis (KATSUYA)

C. Cytology and genetics of Nicotiana and some other plants
51. Cytogenetic studies of the genus Nicotiana XII (TAKENAKA) . . . .
52. Structural changes of chromosomes of Allium scorodoprasum
var. viviparum (TAKENAKA)
53. Selection for high content of nornicotine (FuRusATo and MIYAZAWA)
54. Influence of environment on the fertility of tobacco (FuRusATo)
55. A case of different results from reciprocal crosses: a preliminary
report (LILIENFELD). . . . . . . . . . ..
56: Cytological studies in the genus Polygonum 1. Chromosome

iii

58
59
60
61
62
64
65
66
67
69
70
72

73
74
75
76
77

77
79
80

81

CONTENTS

iv

57.

numbers in the genus Polygonum and related genera (DOIDA)
Developmental studies in the genus Polygonum III (DoIDA) . . . .

D. Biometrical genetics and problems of breeding
58. Biometrical analysis of matroclinal inheritance in rice (SAKAI)..
59. Effect of matroclinal inheritance on the genetical variation ina hybrid population of autogamous plants (lYAMA)..........
60. Biometric-genetical study of seed size characters in cultivatedvarieties of rice (SAKAI and PINTO)
61. Analyses of strains of Drosophila melanogaster selected for
abdominal bristles (YAMADA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
62. Heritability analysis of body weight in purebreds and theircrossbreds in the domestic fowl. (KAWAHARA)..............
63. Viability and susceptibility to a visceral form leucosis in two
purebreds and their reciprocal crossbreds in the domestic fowl
(KAWAHARA)

82
83
86
88
88
90
92

93

E. Mathematical genetics
64.
65.
66.
67.

A formula giving the effective population number based on
the sampling variance in gene frequency (KIMURA).... .. .. ..
Some calculations on the mutational load (KIMURA)
On the substitutional load of a population (KIMURA)
Substitutional load as a measure of gain in genetic information(KIMURA) .... .. .. .. .. .. .. .. . . . . .. .. .. .. .. .. .. . . .. .. . . .. . . ..

96
97
100
101

F.

Biochemistry of pigments and other substances in plants
Genetical studies on the red and yellow eye pigments in
Drosophila (TAIRA)
69. Genetical and biochemical studies on the metabolism of pteridines in Drosophila-The structure of the yellow pigment(NAWA)....................................................
70. Relations between anthocyanin contents and three factors which
condition the quality of flower color in pansies (ENDO) .. .. ..
71. Anthocyanin of purplish blue and deep purple flowers of pansies(ENDO)
72. Genes controlling flower coloration and their dosage effect incompetitive expression (ENDO).. .. .. .. .. .. .. .. .. .. .. .. .. ....
73. Practical value of citbittol A as a reagent in the examinationof human taste (OGAWA)
68.

G. Genetics and biochemistry of microorganisms
74. Subunits of H 1 gene in salmonella (IINo).. .. .. .. .. .. .. .. .. .. ..
75. Unequall recombination in salmonella (IrNO) .... .. .. .. .. .. .. ..

102

104
105
107
107

no
111
112

,

CONTENTS

76.
77.

A relation of metabolism to UV induced mutation in yeast(NAKAI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .
Cytological studies on the nucleus of yeast (YONEDA).. . . . . . . . .

v

113
114

H. Radiation genetics in animals
78. Nature of radiation-induced mutations detected in the silk-worm
by the method of specific loci (TAZIMA and ONIMARU) ..... . 115
79. X-ray induced dominant lethals from irradiation of different
stages of oogenesis in the silkworm (TAJIMA and ONIMURA).. 117
80. Some observations. relevant to the possible cause of radiationinduced sterility in the male silkworm (TAZIMA) . . . . . . . . . . .. 118
81. Recovery of irradiated spermatogonia of the silkworm and thestage limit for the production of functional sperms (SADO) .. 119
82. Duration of spermatogenesis in the silkworm (SADO) .. .. .. .. .. 120
83. Studies on mutation rate by chronic irradiation of mice (SUGAHARA, TUTIKAWA and TAKEDA), .. .. .. .. .. .. .. 121
84. Effect of X-rays on the rate of mitosis (YOSIDA and TAKAHASHI) 123
85. Chromosome breakage at metaphase and fragment formation
at anaphase after X-irradiation (YOSIDA and TAKAHASHI) " .. 124
86. Nature of chromosome bridges after X-irradiation (TAKAHASHI
and YOSIDA) " . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124
87. Relation between micronuclei and chromosome fragments after
X-irradiation (TAKAHASHI and YOSIDA) .. .. .. .. .. .. .. .. .. .. .. 125
88. Transplantation experiments with spontaneous and radiationinduced leukemias in mice (KURITA).. . . . . . . . . . . . . . . . . . . . . . . 125
89. Relation of post-irradiation treatment to radiation-induced mutation frequency in yeast (NAWA and SAITO)................ 127
90. Synthesis of contractile proteins in X-irradiated embryos of
Triturus pyrrhogaster Boie (Ogawa),, . . . . .. 127
91. Heterozygous effects of induced lethaIs and their persistence
in populations (OSHIMA and KITAGAWA) .. "" .. 129
92. Doubling dose as a common unit of measuring mutabilities inpolygenes and major genes (YAMADA) .. . . . . . . . . . . . . . . . . . . . . 132
93. Doubling dose of polygenic characters (YAMADA and KITAGAWA) 133
94. Radiation effects on germ cells of rainbow trout (HARADA, AI,
AI and KONDO) .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135
I. Radiation genetics in plants
95. Comparison of radiation effects of fl- and r-rays on einkorn
wheat (MATSUMURA)........................................ 136
96. Radiation effects of fast and thermal neutrons on wheatA) Genetic effects of neutrons on einkorn wheat (MATSUMURA) 138
97. B) Relation of ploidy to chromosome aberrations (NEZU)
140

CONTENTS

vi

98.
99.
100.
101.

C) Relation between ploidy and radiation effects (MATSUMURA

and NEZU)
Radiosensitivity in polyploid plants (FuJII and MATSUMURA) .. ..
Radiosensitivity in vegetatively propagated plants (MATSUMURA
and FUJII)
Cytological studies on X-rayed rice (HUANG) .. .. .. . . . . .. .. .. ..

142
143
144
145

J. Genetics and cytology of tumors
102.
103.
104.

105.

Variation in the frequency of tetraploid cells in the course oftransplantations of the Yoshida sarcoma (YOSIDA) .. .. .. .. .. 146
Invasive ability of diploid and tetraploid tumor cells of the
Yoshida sarcoma (YOSIDA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
Double inoculation of hyperdiploid and hypotetraploid cell strainsof the Ehrlich ascites carcinoma and their invasion into organs(YOSIDA and ITo)
148
Effects of extract from Gentiana upon living plant tissue (TAKENAKA, OGAWA and ODASHIRO)....
..
..
.. .. .. 149

K. Growth, differentiation and regeneration
106. Effect of glucuronic acid on the growth of the animal embryo(OGAWA)
107. Development of muscle protein in the regenerating limb tissueof Triturus pyrrhogaster, Boie (OGAWA) .. .. .. .. .. .. .. .. .. ..

L. Technical note
108. Invariance of X-ray spectra at various depths in phantom(KONDO and KATO)
109. Silver-phosphate glass dosimeters and their application to X-ray
dose measurements inside X-rayed mice and to separate
measurements of thermal neutron fluxes and gamma dosesin the JRR-1 reactor (KONDO) .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..

150
151

153

155

.,.

GENERAL STATEMENTThe year of 1959 was very important for our Institute because 10 years
have passed since its establishment. On the 1st of June we have celebrated
the 10th anniversary of its foundation. As the weather was fortunately
fine, 281 friends of the institute who contributed directly or indirectory to
its establishment and development attended the celebration, which closed
on a cheerful and hopeful note. In commemoration of the Day, public
lectures in cluding scientific movies were given in Tokyo and Misima.
The number of research reports assembled in this annual report is 145,
which exceeds the figure of last year by 26.
In order to collect rice and related species, five members participating in the studies on the origin of cultivated rice traveled to China, Africa,
India or Burma, where they collected many seeds and specimens of wild
as well as cultivated rice. So far we could obtain 21 species. Five arestill missing from our collection but we are confident that they will be
added in near future.
Dr. T. Tateoka, Dr. S. Sakaguchi and Mr. S. Sakamoto are continuing
their studies at the University of Montreal, Canada, Yale University andMinnesota University, U.S.A., respectively. Dr. Kondo came back after a
14 month study in the U.S.A.
Our institute had a great number of visitors including 28 from abroad.
X-ray laboratory, special silkworm house and a trasferable birdproof
net-cage were newly built.
In addition, a neutron radiation apparatus, a 137Cs r-ray radiation apparatus, an inverted biological microscope, a micro-spectrophotometer anda low temperature centrifuge were obtained.
Dr. M. Kimura received the prize of the Japanese Genetic Society forhis contributions to the knowledge of the genetic population load and its
significance in evolution. Dr. H. Kihara was honored by a prize of Leopoldina, the Germany Academy of Biologists, on the occasion of the 100th
Celebration of Charles Darwin's publication "On the origin of species bymeans of natural selection".

ABSTRACTS OF DIARY FOR 1959
Jan.
Jan.
Jan.

8. Committee meeting of Animal Genetics group.
14. Meeting of Tobacco Research workers.
15. Meeting of all Japan poultry Breeding Association.

2

ABSTRACTS OF DIARY FOR [959Jan.
23. 72nd meeting of Misima Geneticists' Club.
March 11. 23rd Biological Symposium.
March 19. 24th Biological Symposium.
April 17. 73rd meeting of Mishima Geneticists' Club.
June
1. The 10th Commemoration day of National Institute of Genetics.
June
6. Memorial Lecture and movies (in Tokyo)
June
27. 75th meeting of Misima Geneticists' Club.
June
27. Meeting of all Japan Poultry Breeding Association.
June
29. 25th Biological Symposium.
July
17. 76th meeting of Misima Geneticists' Club.
July 22-25. 3rd series of Summer Seminar on Genetics.
Aug.
10. 26th Biological Symposium.
Aug. 24-26. Seminar on radio-isotopes.
Sep.
25. 77th meeting of Misima Geneticists' Club.
Sep.
26. 27th Biological Symposium.
Oct.
18. 28th Biological Symposium.
Oct.
23. 78th meeting of Misima Geneticists' Club.
Dec.
18. 79th meeting of Misima Geneticists' Club.

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

Members
Department of Morphological Genetics:
Yataro TAZIMA, D. Ag., Head of Department
Motoo KIMURA, Ph. D., D. Sc.
Eiichi INAGAKI and Kimiharu ONIMARU
2. Department of Cytogenetics:
YO TAKENAKA, D. Sc., Head of Department
Tosihide H. YOSIDA, D. Sc.,
Yoshiaki YONEDA, Kazuo MORIWAKI and Tsuguo TATEOKA, D. Sc. (inCanada)3. Department of Physiological Genetics:
Chozo OSHIMA, D. Sc. Head of Department
Hitoshi KIHARA, D. Sc., M.J.A., Flora A. LILIENFELD, Ph. D.,
Toshifumi TAIRA, Sadao SAKAMOTO (in U.S.A.) and Koichiro TSUNEWAKI
4. Department of Biochemical Genetics:
1.

STAFF5.

6.

3

Mitsuo TSUZITA, D. Ag., Head of Department
Yoshito OGAWA, M. D., Saburo NAWA, Bungo SAKAGUCHI,
Seizo TSUDA, D. Ag. (in U.S.A.), Toru ENDO and Tetsuo IINo, Ph. D.
Department of Applied Genetics:
Kan-Ichi SAKAI, D. Ag., Head of Department
Hikoichi OKA, D. Ag.,
Yukio YAMADA, Akira MiYAZAWA, Takatada KAWAHARA and ShinyahAMA, D. Ag.
Department of Induced Mutation:
Seizi MATSUMURA, D. Ag., Head of Department
Tsutomu SUGAHARA, M. D.,
Sohei KONDO, D. Sc., Kiyoshi TUTIKAWA and Taro FUJII

Part-time Staff and Research Associates
Kan OGUMA, D. Ag., Ex-Director, Emeritus Professor of Hokkaido Univ.
Yoshimaro TANAKA, D. Ag., D. Sc., M,J.A. Emeritus Prof. of Kyushu Univ.
Taku KOMAI, D. Sc., M,J.A. Emeritus Prof. of Kyoto Univ.
Yoshinari KUWADA, D. Sc., M.J.A., Emeritus Prof. of Kyoto Univ.
Saziro MAKINO, D. Sc., Professor of Hokkido Univ.
Tamaki SHIMAMURA, D. Sc., Professor of Nagoya Univ.
Kazuo FURUSATO.

Research members under the Grant from the Rockefeller Foundation
Rice Research Group:
Tadao KATAYAMA, Mitsuya NEZU, Keizo KATSUYA, Takashi NARISE, Hiroko
MORISHIMA, Kokichi HINATA, Shohachi SHIMOYAMA and Yukio DOIDA
Animal Genetics Group:
Toshihiko SADO, Kazuhiko UTSUMI, Teiichiro TAKAHASHI, Kazuo SAITO
and Osamu KITAGAWA
Sayaka NAKAI

Department of Administration
Kunio SHIMIZU, Head of Department
Sumiyoshi SUGIO, Chief of General Affairs Section
Toichi YOSHIKAWA, Chief of Finance Section
Naomi MATSUBARA, Hiroko NAKANO and Junzo KADOWAKI
Clerks, Typists, Telephone operators, Chauffer, Field Laborers andJanitors

4

RESEARCH PROGRAM FOR 1959

Misima Branch of Hatano Tobacco Experiment Station
Masao TANAKA, Head Assistant...... 5

All Japan Poultry Breeding Association
Hitoshi KIHARA, President
Kan-Ichi SAKAI, Vice-president

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
Seizo KATSUNUMA, President of Nagoya University
Yoshichi ASAMI, Emeritus Professor of Tokyo University
Toshitaro MORINAGA, Director of National Institute of Agricultural Sciences
Hideo MATSUKUMA, President of Japan Monopoly Corporation
Toshio SAITO, Governor of Sizuoka Prefecture
Yakichi NOGUCHI, Professor of Tokyo University
Riichi KAWAKAMI, Head of Department of Biometry, National Institute of
Public HealthToshinobu ASAI, Professor of Tokyo University
Takeo SAKATA, President of T. Sakata Company
Ichiro ISHIKAWA, Commissioner of Atomic Energy Commission
Daigoro MORIWAKI, Professor of Tokyo Metropolitan University
Kunizo FUKUDA, Emeritus Professor of Tokyo University
Kenpo TSUKAMOTO, Director of National Institute of Radiological Sciences

RESEARCH PROGRAM FOR 1959
Department of Morphological Genetics
Genetics of silkworm (TAZIMA and INAGAKI)
Studies on the food preference in silkworm (TAZIMA)
Radiation mutagenesis in silkworm (TAZIMA and ONIMARU)RESEARCH PROGRAM FOR 1959

5

Genetic effects of chronic r-irradiation upon silkworm (TAZIMA and ONIMARU)
Theoretical studies of population genetics (KIMURA)

Department of Cytogenetics
Cytology and genetics of tumors (Y osIDA)
Experimental breeding and genetics in mice and rats (Y OSIDA 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 Prumns yedoensis (TAKENAKA)
Cytological studies on the yeast cell (Y ONEDA)

Department of Physiological Genetics
Genetical studies on insecticide-resistance of Drosophila (OSHIMA)
Physiological studies on eye-pigment formation of Drosophila (TAIRA andOSHIMA)
Population genetics of deleterious genes in natural populations of Drosophia (OSHIMA)
Studies on the origin of wheat (KIHARA)
Studies on substitution of nucleus in wheat and related species (KIHARA)
Right- and left-handedness in plants (KIHARA and LILIENFELD)Studies on stoneless pomegranates (KIHARA)

Department of Biochemical Genetics
Biochemical genetics of insects and microorganisms (TSUJITA, NAWA andIlNo)
Embryological and biochemical studies of silkworm (TSUJITA and SAKAGUCHI)
Biochemistry of the mechanism underlying variations in flower colors inplants (ENDo)
Biochemical studies on the differentiation of muscle protein in animals(OGAWA)
Biochemical studies on the mechanism of cell division in animals (OGAWA)
Biochemistry of bitter substance in Citrullus colocynthis (OGAWA)
Studies on the fine structure of gene (TSUJITA)
Genetics of virus (TSUJITA)RESEARCH PROGRAM FOR 1959

6

Department of Applied Genetics
Studies on breeding and genetics in poulty (YAMADA and KAWAHARA)
Quantitative genetics in Drosophila (YAMADA)
Theoretical studies of plant breeding (SAKAI)Studies on competition in plants (SAKAI and IYAMA)
Population genetic studies of 'Red-Rice' growing among upland rice (IYAMA)
Genetic studies on 'cherry-red leaf' in tobacco plants (SAKAI and lYAMA)
Genetic studies on the migrating activity in Drosophila (SAKAI, lYAMA andNARISE)
Polyploidy and sterility in fruit plants (FURUSATO and MIYAZAWA)
Genetical studies on cytoplasmic inheritance in plant (SAKAI and lYAMA)
Genetic studies of some physiological and agronomic characters in rice (OKA)

Department of Induced Mutation
Studies on radiation-protection in animals (SUGAHARA)
Radiation genetics of mice (SUGAHARA and TSUTIKAWA)
Relation between the quality of radiations and mutations (MATSUMURA and
KONDO)
Radiation genetics of wheat (MATSUMURA, FUJII and NEZU)
Radiation genetics and its practical application (MATSUMURA and FUJII)Radiosensitivity in plants (FUJII)
Radiation dosimetry (KONDO)
Triploidy breeding of sugar beets (MATSUMURA)JOINT RESEARCHES SUPPORTED BY THE
GRANT FROM THE ROCKEFELLER
FOUNDATION
1.

Origin of Rice

Section 1. Collection and preservation of Oryza species (H. KlHARA)
a. Strains so far collected are more than 3,000, in which 20 different
species are included.
b. Collection-tours were made by Dr. H. Kihara to Silkkim and Assam,
India, by Mr. K. KATSUYA to Burma, and by Mr. K. FURUSATO to
Africa, in this year.
Section 2. Morphology and physiology of Oryza species (S. MATSUMURA)
a. Comparison of radio-sensitivity among Oryza species (T. FUJII)
b. Genome-analysis of Oryza species (M. NEZU)JOINT RESEARCHES SUPPORTED

7

c.

Susceptibility of wild and cultivated rice strains to blast fungus (K.
KATSUYA)
d. Surface structure of lemma and palea in Oryza species (H. KIHARA
and T. KATAYAMA)e. Investigation of photoperiodic responses in Oryza species (T. KATAYAMA)
Section 3. Population-genetics in wild and cultivated rice (K. I. SAKAI)a. Estimation of genetic variability among and within populations ofwild rice (K. I. SAKAI, S. lYAMA and T. NARISE)
b. Estimation of the percentage of out-crossing by a biometrical method(K. I. SAKAI, S. lYAMA and T. NARISE)
c. Competition between wild and cultivated rice strains (K. I. SAKAI and
T. NARISE)
d. Variation studies of blast disease resistance in wild rice populations(T. NARISE and K. I. SAKAI)
Section 4. Genetic studies in wild and cultivated rice (H. I. OKA)
a. Statistical-systematic studies of Oryza species (H. MORISHIMA and
H. I. OKA)
b. Survey of variations between O. perennis and O. sativa f. spontanea(H. MORISHIMA, W. T. CHANG and H. I. OKA)
c. Crossing-experiments for the sterility of hybrids between wild andcultivated rice strains (K. HINATA and H. I. OKA)
d. Investigation of floating habit in wild Oryza species CH. MORISHIMA
K. HINATA and H. I. OKA)
e. Introgressive hybridization between wild and cultivated populationsof rice (H. I. OKA and W. T. CHANG)
Section 5. Cytogenetics of Ory za species (Y. T AKENAKA)
a. Karyo-type analysis of Oryza species (S. SHIMOYAMA)b. Comparative observation of chromosomes in haploid plants of Oryza
species (C. H. Hu)
c. Embryological studies in Oryza "lpecies (Y. DoIDA)

II. Studies on genetic effects of radiation on animals
Section 1. Radiation mutagenesis in silkworm (Y. TAZIMA)
1. Nature of radiation induced mutations detected by specific loci methodin silkworm (Y. TAZIMA and K. ONIMARU).
2. X-ray induced dominant lethaIs from irradiation of different stagesof oogenesis in silkworm (Y. TAZIMA and K. ONIMARU).
3. Some observations relevant to the possible mechanism of radiationinduced sterility in male silkworm (Y. T AZIMA).
4. Cytological studies of radiation-induced sterility in the male silkworm(T. SADO).

8

JOINT RESEARCHES SUPPORTED

5.

Recovery of irradiated spermatogonia of the silkworm and the stage
limit for the production of functional sperms (T. SADO).
Section 2. Mutation in mammals (T. SUGAHARA).
1. Studies on mutation rate by chronic irradiation of mice (T. SUGAHARA,
K. TUTIKAWA and Y. TAKEDA).
2. Dose distribution inside mice exposed to X-rays (S. KONDO)
Section 3. Cytology and cancer problems (T. H. YOSIDA).
1. Effects of X-rays on the rate of mitosis (T. H. YOSIDA and T. TAkAHASHI).
2. Relation between chromosome breakage at metaphase and fragment
formation at anaphase after X-irradiation (T. H. YOSIDA and T. TAKAHASHI).
3. Nature of chromosome bridges after X-irradiation (T. H. YOSIDA and
T . TAKAHASHI).
4. Relation between micronuclei and chromosome fragments after Xirradiation (T. H. YOSIDA and T. TAKAHASHI).
Section 4. Biochemistry of radiation mutagenesis (M. TSUJITA).
1. Studies on the mechanism inducing mutations after irradiation (S.
NAKAI).
2. Effects of irradiated RNA components on mutation (T. IINo).
3. Relation of post-irradiation treatment to radiation-induced mutation
frequency in yeast (S. NAWA and K. SAITO).
Section 5. Mutations in populations (C. OSHIMA).
1. 1. Heterozygous effects of induced lethals on pre-adult viability (C.
OSHIMA and O. KITAGAWA).
2. Doubling dose of polygenic characters (Y. YAMADA and O. KITAGAWA)
3. The effect of X-ray irradiation on selection response (0. KITAGAWA)FOREIGN VISITORS IN 1959

9

FOREIGN VISITORS IN 1959

"

Feb.
II.
March 10.
March 24.
March 29.
June

June
June
June
Sep.
Sep.

Sep.
Nov.
Nov.

Prof. F. J. RYAN (Columbia University, U.S.A.)
Associate Prof. B. S. STRAUSS (Syracuse University, U.S.A.)
Dr. J. J. MCKELVEY Jr. (The Rockefeller Foundation)
Dr. I. S. BALKE (Minister of Atomic Energy, West Germany)
Dr. H. SAVES
4. Dr. Chung Fu CHENG
Dr. Keh Min LIN
Dr. Hsuin shwen CHANG
Dr. Cheng I LIN
Dr. D. M. QUAN
Dr. T. L. COPLEY
15. Dr. Miss D. PARKER (Librarian: the Rockefeller Foundation)
26. Mr. A. K. M. HUSSEIN (Pakistan)
29. Dr. H. H. SMITH (IAEA, Vienna)
3. Prof. H. KAMENOTO, Prof. R. A. HAMILTON (University of Hawaii,
U.S.A.)
19. Prof. P. N. MEHRA (Botany Dept., Punjab University, India)
Dr. A. J. MACLEED (Federal Dept. of Veterinary Research vom.
Nigeria)
22. Mr. I. P. GUNAWARDENA (Dept., of Agriculture, Ceylon)
Dr. G. P. ARGIKAR (Bombay Agricultural Department, India)
10. Dr. K. HOFMANN (Univ. Kid. Germany)
17. Prof. A. B. BURDICK (Purdue University, U.S.A.)
Dr. H. SLATIS (Argonne National Laboratory, U.S.A.)RESEARCHES CARRIED OUT IN 1959
A.

1.

GENETICS, BIOCHEMISTRY ANDCYTOLOGY OF INSECTS

Studies on the E and U allelic genes in the silkworm,
with special reference to the attachment form between
the Vlth and XIVth chromosome(By Mitsuo TSUJITA)

It is known that under natural condition one end of chromosome VIthand one end of chromosome XIVth are occasionally attached to one another.
On the former chromosome the E complex locus (E region) and on thelatter chromosome the U complex locus (U region) are located. These twocomplex loci effect somewhat similar phenotypes. It has been observedthat in the spermatocytes of the hybrid between domesticated (Bombyxmori) and wild silkworm (Bombyx mandarina) a trivalent made up of two
chromosomes of the former and one chromosome of the latter appears.
Table 1.

Segregation from the Cross Nl2ENe/+ + X +.

====.==-!-=-==--=-.=-:::--::==-------=::-=-=------==::-c---c~·_=__'__I---==-==

Total of 1() batches !

No.
-----

1
2
3
4
5
6
7
8
9
10

,

,

,

Total i(mean)'-:-..= .

---~--=:....:==--=.-:.:=

+

ENe

Nl2

Nl2 ENe

Total

1720

o

o

1573

3293

Table 2.

Segregation from the cross + x Nl2ENe/+ +.

+

Nl2

ENe

Nl2ENeTotal

C.O.V.

132
98
87
113
101
117
128
98
47
49

130
47
58
93
70
91
103
53
44
45

97
78
67
91
82
100
88
63
53
50

109
78
67
94
87
98
114
92
56
52

468
301
279
391
340
406
433
306
200
196

48.5
41.5
44.8
47.1
44.7
47.0
44.1
37.9
48.5
48.5

970

734

769

847

3320

45.3

RESEARCHES CARRIED OUT IN 1959

11

In the present experiment the following subjects were studied. i) Attachment form of the two chromosomes. ii) Eventual relationship between E
and U regions. iii) Relationship between the attached chromosomes andthe trivalent which is found in the hybrid between the domesticated andthe wild silkworm.
From the cross between a female of the strain with attached chromosomes E NeNl2 and a normal male, only two types, Nl2Eve and +, segregated, and from the reciprocal cross four types, N12, ENeNI2, ENe, and +segregated. (Tables 1 and 2)
As shown in the above table, the apparent crossing-over value is 45.4.
It seems, however, that this is not the real value for the following reason:
In a small number of spermatocytes of the male from the cross + X NZZENe!
++, 27 chromosomes can be observed, one of them consisting of two attached chromosomes, but in most of them 28 chromosomes are counted, as
shown in the following table.
Table 3.

Chromosome numbers in the spermatocytes of the malefrom the cross +xNI2ENe/++.

As shown in the above table, among 164 spermatocytes at the firstmaturation division, 30 had 27 chromosomes. On the contrary, among 146
spermatocytes at the second maturation division, 14 had 27 chromosomes.
Thus, the number of the spermatocytes having the attached chromosomes
is smaller in the former than in the latter division. It seems from thisobservation that the attachment of the VIth to the XIVth chromosome in
the male germ cells is unstable. The number of male germ cells in which
both chromosomes be- have as one up to the final maturation division
amounts to about 10% of the second spermatocytes, while in 90% they
maneuver independently, so far as our observation goes.

RESEARCHES CARRIED OUT IN 1959

12

Thus, the crossing-over value shown in Table 2 cannot be taken as thelinkage distance between ENe and N/2, because there are two modes of
behavior of the VIth and XIVth chromosomes at spermatocyte division; in
one of them the attached VIth and XIVth chromosomes behave as one
chromosome, while in the other they do not attach and each behaves as
an independent chromosome.
Further, the ratio of the two types, N/2 and ENe, to the total numberof four types, +, N/2, ENe, and ENeNl2 from the cross + xENeNf2/+ +should be different according to the form of the attachment, as shown in
Fig. 1.

=1-----EN.

(1)

42

(2)

Fig. 1.

Based on these to forms of the attachment, crossing-over value wascalculated.
In the case of the attachment form (1) 50xO.9+4.2xO.1=49.2
In the case of the attachment form (2) 50xO.9+8xO.1=45.8
The crossing-over value shown in Table 2 is 45.3. Judging from thisvalue there is much stronger evidence for form (2) than for form (1).
Moreover, in most spermatocytes of the hybrid between the domesticated,,"",,: i \':.

Photograph 1. Chromosomes is thefirst maturation division of thehybrid between the domesticated(the strain having the attachedchromosome) and the wild silkworm. 27 chromosomes can beobserved. (x 1600)

Photograph 2. The same material
as in photograph 1. 26 chromosomes can be observed. An arrow indicates the attached chromosomes. (x 1600)

RESEARCHES CARRIED OUT IN 1959

13

(the strain having the attached chromosomes) and the wild silkworm, 27
chromosomes were observed, but in a small number of spermatocytes, 26
chromosomes, one of which consists of two attached chromosomes, arefound (Photos 1 and 2).
This indicates that there is no relationship between the attached chromosomes and the trivalent appearing in the spermatocytes of the hybrid between the domesticated and the wild silkworm.
The fact that the VIth and XIVth chromosomes attach suggests an
intimate relationship between them. Furthermore, as the present experimental results show, attachment form (2) is the more probable, placing
the U and E complex loci relatively near to each other. Therefore, some
important relationship between the two regions may be assumed.

2. Genetic and biochemical studies on the new white-egg
mutant in the silkworm(By Mitsuo TSUJITA)The new mutant strain of the silkworm, wax, which exhibits a conspicuously transparent larval hypodermis and white eggs was obtained by X-ray
treatment in 1954.
The gene wax belongs to the Xth linkage group, and crossing experiments
show it to be allelic to WO l on chromosome Xth.
Amounts of uric acid and isoxanthopterin produced in the hypodermis oflarvae homozygous for wo x, w S , WO l and of those of their hybrids were
measured by a paperchromatographic method. In order to ascertain theamount of lost uric acid and isoxanthopterin in the procedure of measurement, definite amounts of the two substances were absorbed in a filter
Table 1.

Strain~._----

Isoxanthopterin (p.gjg in dry matter weight, after
paperchromatography)
II

III

Mean

975

938

938

950.33

wS

188
375

150
375

163
370

167.00373.33

wax

trace

trace

trace

trace

WSxwo x

263
75
1010

225
113
938

338
113
1013

275.33100.33987.00-+ (Japanese 501

X Chinese 501)

wo l

WOlXwo x
w1Xwo x

14

RESEARCHES CARRIED OUT IN 1959

paper. The amounts extracted from the spot appearing on the paper
were measured. The remainder, initial amount-extracted amount, may
be considered as lost in the procedures. The results obtained with thelarval hypodermis were corrected accordingly.
The corrected amounts of uric acid and isoxanthopterin are given in the
Table 1 and 2.
Table 2.

----Uric acid (mg/g. in dry matter weight, after
paperchromatography)

Exp. No.

I

II

Strain(JIa~~~-o:-~e-es-~O-~-O-I)- ,------:~~:-~-~-~ :---8-:-:-:-:-r

III

Mean

: ::- - ::99: - -

1

w3

81.00

63.75

69.00

wax

trace

trace

trace

trace

21.75
I
9.75
87.00
~_ _
1_~

26.25
6.00
73.50

22.50
8.25
85.50

23.50
8.00
82.00

w 3xw ax
WO!xwo x

~x wo

x

I

-'------

71.25---'--------------- - - - - - - -Tables 4 and 5 show that the amounts of isoxanthopterin and uric acidin the hypodermis tissue of larvae homozygous for wax are much smaller
than in the larvae homozygous for wo! or w 3 • In the case of uric acid incomparison with isoxanthopterin, a large part is apt to be lost in the experimental procedure, so it is a matter of prime concern that the accurate
amount of uric acid cannot be ascertained by paperchromatography. Therefore, in order to obtain more exact results, a method using uricase wasapplied, and the experimental data are given in Table 3.
Table 3.

Uric acid (p.gjmg. in dry matter weight, after uricase method).

------- = Genotype I
1
2

Mean

aa

ad

Exp. N~--­____.. __i_.
0.25
0.23
0.24

2.5
2.6
2.6

24.3
23.1
23.7

25.6
26.1
25.9

+
108.2
106.5
107.4

The table shows that the amount of uric acid in the hypodermal tissueof the wax homogeneous larvae is only 1/100 of that found in the WO l larvaeand 1/10 of that found in the od larvae, which was hitherto assumed tohave the smallest amount among the known mutant strains with transparent larval hypodermis.

15

RESEARCHES CARRIED OUT IN 1959

Thus, it is characteristic of the W°'" mutant its amount of uric acid andisoxanthopterin are much smaller than in any other known mutant strain
with transparent larval hypodermis.
It was found from in vitro experimental results that the activity ofpterin dehydrogenase in the larval hypodermis of the mutant is somewhat
stronger than in the normals. Therefore, it seems that there is nothingabnormal with the enzyme itself. In order to clarify the action of wO'",
a small amount of 2-amino-4 hydroxypteridine (AHP) was injected into thebody-cavity of larvae homozygous for w°'" and the amount of isoxanthopterin
produced in the hypodermis was periodically measured after 1, 2, 3, 4, 14,
20, and 24 hrs. The experimental results are shown in the following table:
Table 4.

Change of the amount of isoxanthopterin in the hypodermal
tissue after injection of AHP into
the larval body cavity.

Genotype
Duration -~-~_________after injection --------_
Control
1 hr.
2 hrs.
3
4

14

19*

28
29
30
31
16

20
24

18

40*
49
73

69
69
65
64
45
- - ------------* Relative amounts
As the table shows, the amount of isoxanthopterin in the hypodermis of
the larvae homozygous for w°'" increases during the first 4 hours after the
injection of AHP, and then decreases rapidly, showing the initial amount
14 hours after the injection. On the contrary, the amount of isoxanthopterinin the hypodermis of the larvae homozygous for wol increases in the courseof the first 3 hours after the injection and then decreases very slowly. The
normal amount of isoxanthopterin appears 24 hours after the injection.
Thus, it takes more time for wol than for w°'" larvae to regain the initialamount.
It may be assumed that isoxanthopterin and uric acid newly produced in
the hypodermal tissue penetrates into the body cavity passing through cell
membranes and that the permeability of the substances is controlled byw°'" or w nl •

16

RESEARCHES CARRIED OUT IN 1959

3.

XRay induced Nl-type mutants of the silkworm(By Mitsuo TSUJITA)

Female moths homozygous for HKp whose pupae were treated with 8000r
X-rays were mated with normal male moths. Many Nl-type mutants(non-lunar silkworm larvae) were obtained from these hybrids. The resultsof the experiments which were carried out during the years 1954-1958 aresummarized in the following table.
Table 1.

Nl-type mutants obtained from 1954-1958.

Year

RearingseasonNo. ofexaminedbatches

1954
1955

SummerSpring

167
230

Total
-----

- ~ - - - - - - -

13
16

2
20

3
3

15
26

1955

Summer

31

2

1

0

1

1956

Summer

150

20

23

3

1958

Summer

15

10

2

35
58

100

33

7346

65
4

29568

7379
29633

204

208

81

25657

25738

85

15682

15767

_ _ _
1
_ _ _ _--------

Since examination for exceptional types was done in the early larvalstage, it seems likely that a part of the Nl-type mutants were not detected.
Therefore, the total number of the Nl-type mutants may be actually larger
than the number shown in the above table.
There are two kinds of Nl-type mutants. Larvae with the genotype
N[1j + show almost the same characteristics as those with the genotype
Nll + in that they both lack the lunar patterns on the 5th segment andthe star-shaped markings on the 8th segment, and in the presence ofmodifiers, rudimentary patterns appear on both segments. However,larvea
with the genotype Nt'! + are different from those with the genotype Nl/ +or N[1j +; they completely lack lunar markings, but in certain genetic
background, star-shaped markings appear. Furthermore, they lack the
caudal horn on the 11th segment.
Two Nl! type mutants obtained in the spring of 1955 were crossed withthe odk strain and crossing-over values were calculated. The results were
4.6 and 5.2, respectively, while the crossing-over value between Nl2 andodk was 7.2.
Three Nl! type mutants and 2 Nl2 type mutants, which were obtainedin the experiment of 1958, were examined. Two types segregated fromthe cross between the N[1 mutants and the oa strain, namely, N[1 larvae
with transparent hypodermis and normal larvae with non-transparent hypodermis. And from the cross between the Nl2 mutants and the oa

17

RESEARCHES CARRIED OUT IN 1959

strain, segregation of two types, Nl2 larvae with transparent hypodermis
and normal larvae with non-transparent hypodermis, was observed.
The crossing-over value between the new NlI and odk was 3.2 and thelinkage distance between the new Nl2 and odk was 6.3, as shown in thefollowing tables.
Segregation from the cross odk X odk/NlI.

Table 2.

NlI--- ---------

Phenotypes+~ __odk_ ~~_,_O~i

Batches
10 batches

37
Table 3.

~~~pes I-~

Batches

1122

1014

37

-------'C.O.V.
1_'

2210

_

3.4

Segregation from the cross odkxodk/Nl2.
+N1

2

Nl2
Total

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

-

+O<Zk

odk

+Odk

odk

109

1433

1294

73

-_.-~-.

10 batches

i

Total

i

C.O.V.

I

290-9-11~3

From the crosses +/NlI x Di/Di and +/Nf2 x Di/Di, respectively, NlI and
Nf2 larvae and normals segregated. These larvae exhibited slightly" dirty"
larval marking characteristic of Di.
From the experimental results above-mentioned it might be inferred thatin all of these mutants a deficiency was involved in the portion extending
from the U region to the marker odk of the 14th chromosome, and thatthe extent of the deficiency is not always the same.
A spontaneous Nl-type mutation occurs very rarely. However, it may
be said from the present experiment that the frequency of the mutation
increases strikingly under irradiation with X-rays.

4.

Genetic maps of the eighth linkage group of the silkworm(By Yataro TAZIMA, Tamiji OZAWA, Eiichi INAGAKI and Takao KOBAYASHI)

In his classical paper on amylase activity of silkworm larvae, MATSUMURA(1934) reported that there are two different genes, ae and be, which control
the activity of enzymes in the digestive juice and in the body fluid respectively, and that they belong to the same linkage group. According to him,
recombination value between these two genes is 1.1%. This group was
temporarily called the eighth group, according to the order of discovery,
though it was not certain whether the group is actually a new one or not.
Since then nobody has tested the linkage relation of ae and be with otherRESEARCHES CARRIED OUT IN 1959

18

morphological genes, until a joint survey of silkworm linkage groups wasundertaken on an extensive scale in 1955.
As a member of the committee, the senior author was assigned to carryout the survey of this group. He confirmed its independence from thealready established 14 groups.
In 1956 one of the authors, Ozawa, found that the st gene (stony, a larval character) belonged to this linkage group among a number of othergenes tested with ae. Recombination values of st-ae and st-be were
5.7% and 11.4% respectively, which indicated the order of these three
genes to be st-ae-be, though the values themselves were not reliable
enough.
In order to determine the exact loci of these genes, we undertook threepoint tests. The Indian polyvoltine race, "Pure Mysore ", which was the
only triple dominant strain among the many tested, was outcrossed to the stae be stock. F 1 males were backcrossed to triple recessive females. However, it was found that the activity of the +be gene of "Pure Mysore"
was too weak to confirm the amylase action in heterozygotes. Therefore,+stae+!Jejst+aebe individuals were built up by using other strains, Hinode(+ae+), Taishohaku (+ + be) and Stony (st ae be). These triple heterozygotes were backcrossed to st ae be. Recombination values obtained ,vere
as follows:
st-ae, 2.8%; ae-be, 1.4%; st-be, 4.2%.
From those data, the linkage map of the eighth group may be represented as:st(O.O) -ae(2.8) - be(4.2).

5.

Structure and function of pleiotropic genes of the silkworm IfI.
Genetical study of four mutant strains induced
independently from the EFil gene
(By Yataro TAZIMA and Eiichi INAGAKI)

The pleiotropic effect of genes of the E pseudo-allelic group suggeststhat the E-region might be a complex of several closely linked component
subunits having different functions. The most reliable method of examining
this supposition is to test recombination between those subunits. However,no marker genes contiguous to the E-region have been found, whichmakes the recombination test inapplicable. Therefore, approaches weremade by inducing mutations at the supposedly specific sites which might
correspond to components of a compound character.
Up to the present, we have obtained a large number of presumed
mutants for each component. In this report reference will be made only

19

RESEARCHES CARRIED OUT IN 1959

to the information concerning four mutant strains obtained from the complex gene EFJl. The characteristics of those strains are shown in Table 1.
Table 1. Characteristics of four mutant strains as represented by the
obliterating effect on components of compound characters.
-----~~~-_~c__ ==~-_===~==~~-~-=====-=-~~~~===

Obliterating effect in terms ofpenetrance
StrainRepresentative
phenotype4th segment

5th segment
I-

Markings I------

Legs-------'----

Elm
El 22

El-1
El-4

0.14
0.51

Elas
EI 131

E 1-6

1. 00
1. 00

1.00:
*
**

-

Le;;-I Markings

E l-6

I
I'

0.92
0.93
1.00
1.00

I

II

0.00
0.00
0.60
0.41

0.29
0.29
0.09*
1.00**

100% disappearance in individuals with extra markings or legs.
legs of 5th segment considerably degenerated.
legs of 5th segment completely degenerated.

Even though the penetrance varies in the same mutant strain according
to the component trait, specificity of each strain is clear so far as the siteand magnitude of change are concerned. Representative phenotypes ofeach mutant strain are given in the second column of the table. Thesemutant strains are maintained by backcrossing to the normal and yields
several types of pheno-variants in every generation. For example, whentype El-l of the EI 122 strain is crossed to the normal, pheno-variants El-1
(26.3%), El-2 (13.3%), El-4 (5.9%) and quasi-P (1.0%) appear (for typeclassification, refer to Ann. Rept. Nat. Inst. Genetics No.9, p. 11). This
fact suggests that all these types might be modified expressions of one
mutant gene, presumably derived from Efill.
Each type was then backcrossed to the normal to confirm the differencesin phenotypic expression. The results are given in Table 2.
Even in the same strain considerable differences in expression wereobserved according to the type of parents used for the cross. For instance,
within the EI 122 strain, disappearance of additional crescent markings on
the 4th segment was more extreme in the effspring of quasi-P than inthose of El-1 or El-2. The obliterating offect on extra-abdominal legs on
the 4th and 5th segments was more extreme in the offspring of El-2 thanin those of El-1. The similar drift in phenotypic expression was also
observed between the different phenotypes of Eh2 strain. It may be inferred, therefore, that the cause of phenotypic modification within the samestrain is due to modifiers.

20

RESEARCHES CARRIED OUT IN 1959

Thus, two characeristics are distinguished, one is proper to the strain
and the other is variable within the same strain. The former seems to becontrolled by a mutant gene specific to each strain while the latter is under
the control of non-specific strain modifiers.
Table 2.

The results of testcrosses of different phenotypes of mutant
strains to normals.
Obliterating effect in terms ofpenetrance

1

StrainI
Type ofBreeding I
parent usedseasonfor cross4th segment- - - - - - _ .. "Markings

5th segment··L~I Markings

--_.-

El-l
EI-2
quasi-P*

EI l22
EI l22
EI l22
El 22
EI 22

El***
EI-4

Legs

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

59-1
59-1
59-3
59-3
59-1

0.14
0.49
0.59
0.22
0.51

0.00
0.00
0.00
0.00
0.00

0.92
1.00
0.99
0.85
0.93

0.29
0.39
0.17**
0.11
0.11* Phenotypically almost similar to P (Normal) except for the traces of extra legs;considered to be a modified form of El!22'** Lower penetrance might be attributed to a difference in environmental conditions.
*** Abbreviation of " EMl".
Table 3.

Modifications of phenotypic expression in the offspring ofdifferent phenotypes crossed to normals------- - - - - ------Obliterating effect in terms ofpenetrance
Strain andItype of parent,

Incubation
temperature-------

4th segment
Markings

Legs

----

- _...... - - - -5th segment
Markings

Legs. _ _ _ _ _1..

EI l22El-l
EI l22
EI-2
EI l22
EI-4

30°C
20°C
30°C
20°C
30°C
20°C

0.12
0.15
0.16
0.16
0.71
0.70

1.00
0.92
1.00
0.97
1.00
0.99

0.00
0.00
0.00
0.00
0.00
0.00

0.33
0.03
0.15
0.05
0.12
0.00

EI"
El-l
EI 22
El-4

30°C
20°C
30°C
20°C

0.41
0.38
0.61
0.52

0.98
0.86
0.99
0.82

0.00
0.00
0.00
0.00

0.00
0.00
0.01
0.00

Elsa
El-6

30°C
20°C

1.00
1.00

1.00
1.00

0.03
0.86

0.75
0.65

EI 13l
El-6

30°C
20°C

1.00
1.00

1.00
1.00

0.07
0.77

1.00
1.00

RESEARCHES CARRIED OUT IN 1959

21

Since it had been known that incubation temperature affects the expressivity of some gene controlled characters, the effect of incubation temperature upon the expression of the mutant traits was tested by subjecting theeggs to high or low temperatures. The results are shown in Table 3.
The response pattern to extreme temperatures differed according to themutant strain but was rather similar among the different types of thesame strain. This result supports the above interpretation that the different types in the same strain represent modified phenotypic expressions of
the same gene.
From these observations, it may be assumed that the four mutant strainsused in the present experiments might have been the products of mutation
at different sites. If this is true the EEl gene may be understood as being
composed at least of four sites which can mutate independently.

6. Genetic variations in DDT-resistance of laboratory strains
and natural populations of Drosophila melanogaster.
(By Chozo OSHIMA)

The level of DDT-resistance of twenty laboratory strains of D. melanogaster
Table 1.

Mortality of laboratory strains observed 24 hours after exposure
to 4% DDT test paper and their LD50s and slops.

-------

Exposure time
(Hours)Laboratory strain

I

LD50

Slope

89.7
73.0
84.2
49.8
58.8
78.0
42.3
89.9
19.6
68.0
55.1
46.4
73.9
33.1
45.8
69.2
45.1
99.1
74.7
58.9

9.4
11.4
9.6
16.2
13.2
11.4
17.2
10.2
26.8
12.2
15.0
17.6
12.0
21.8
18.8
12.6
19.2
8.4
12.4
14.4

7.19
6.53
6.29
1.96
4.29
7.81
5.19
8.88
2.33
6.39
5.80
3.63
7.43
3.56
3.46
7.10
2.59
7.00
8.48
6.60

62.7

14.49

5.63

I

8 Mortality

16 Mortality I

(%)

(%)

OsyoroOtaru
SapporoToyamaKatsunuma
Asitakayama
Hatijyozima
Anjyo
HikoneKyoto
Hirakata
MiyazuTateba-sima
Kama-sima
Iguro-sima
Koti
UwajimaHikosan
Kirisima
Ao-sima

32.3
20.8
33.9
34.1
24.5
15.5
0.8
18.9
1.0
16.9
8.7
17.4
14.5
4.6
18.1
12.4
24.4
43.1
6.9
6.1

Overall average

17.7

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20

I

RESEARCHES CARRIED OUT IN 1959

22

collected from different localities in Iapan and maintained by mass-mating
for several years was tested by exposure to 4% DDT test paper preparedby the WHO in Geneva. The level of DDT-resistance of F 3 flies originatedfrom many female flies captured in nine natural populations was tested
also by the same method.
The LD50 and slope of the dosage-mortality regression line of thesestrains were obtained from mortality records 24 hours after exposure to
two time-determined dosages. These results are shown in Table 1 and 2.
Table 2.

Mortality of natural populations observed 24 hours after exposure
to 4% DDT test paper and their LD50s and slopes.
==~~=-===~

Exposure time
(Hours)Population

I!

8 Mortality

16 Mortality I

(%)

(%)

38.4
54.8
43.2

75.7
91.9
81.0

-li-.3-'

67.4

16.9

73.3

LD50

Slope

9.6
7.7
8.8

4.66
4.63
4.72

13.0
11.8

7.01
7.05

14.0
20.4
24.0
16.6

4.85
3.66
3.04
4.50

13.99

4.91

I

Suyama-1
Suyama-2
Suyama-3
Juriki-1
Juriki-2~I- - - - - - - - - - - · · - - 1I

Kofu
Katunuma-1
Katunuma-2
Katunuma-3
Overall average- ~ ~ - -

17.9

___+-

II

56.7

I

.~_~l~:L. _}: _~I
23.0

62.3

1

The difference between the two overall average LD50s of laboratory
strains and F 3 flies of natural polulations was not as large as might havebeen expected considering the anticipated effect of extended laboratory
culturing without selection in the former.
A positive correlation between slope and population size was expectedon the basis of the original populations of some strains. This was verified
in natural populations which were collected by the authors in October, 1959.
There was a significant negative correlation (r= -0.63) between slopesand LD50s. Strains having flat slopes were, in general, more resistant
than those with steep slopes.

RESEARCHES CARRIED OUT IN 1959

23

7. Frequencies of deleterious chromosomes in natural populationsof Drosophila melanogaster.
(By Chozo OSHIMA and Osamu KITAGAWA)

Many flies were collected from natural polulations at three places in
Suyama and two places in J uriki in Shizuoka Prefecture and one place in
Kofu and three places in Katunuma in Yamanashi Prefecture in October
1959. The second chromosomes of male flies were isolated individually by
the CMI method.
The mating scheme of the CMI method.

+ +,- +---+ +--T

M-5 Cy Ubx---¥¥x:--------~~

Samarkand'-Pm SUI~\,

P,",,- Ebx ~~M-5 J1y Ubx ¥¥ X /

+ + +

+ +

I

+Cy __+~\,'+-Pm + ¥¥x~

+

Cy

+

Pm + -~~CB

M-5 Cy Ubx

M=SPm Sb ¥¥x'-!

@:

A single male fly of the flies collected from natural populations was crossed

24

RESEARCHES CARRIED OUT IN 1959

to female flies having inversions and dominant marker genes on eachchromosomes. The M-5, Pm and Ubx heterozygous male fly was crossed
to female flies having Cy, Pm marker genes and inversions on the second
chromosomes and homozygous background. In the following generations,
female and male flies heterozygous for the Cy chromosome and for oneof the chromosomes obtained from natural populations were mated. If
the wild-type second chromosome had deleterious genes, the proportion ofwild type flies in the next generation would be decreased below 0.33.
When a number of chromosomes were tested by the above method, aconsiderable number of lethal, semi-lethal and deleterious second chromosomes were detected. The frequencies of lethal and semi-lethal chromosomes found in each natural population are shown in Table 1.
When the data are compared with that reported by several other investigators, we find that the frequencies of lethal and semi-lethal chromosomes in Japanese natural populations are somewhat than those in
American and Israel populations and almost equal to those in Korean
populations. The natural populations of Suyama and Juriki were small.
The natural populations of Kofu and Katsumuma were large and coexistingwith other species, D. lutea, D. hydei and D. immigrans. The causes forlower frequencies than expected in Suyama and Juriki populations arepresumed to be associated with conditions with make most of the lethal
Table 1.

Frequencies of lethal and semi-lethal second chromosomes
in natural populations.

---.--

Population
I

Suyama-l
Suyama-2
Suyama-3

No. of chromo No. of lethal
testedchromosomes

No. of semilethal chromo

%

43
19
22

4
2
3

I

84

9

10.n-1

Juriki-l
Juriki-2

43
27

5
2

Total

70

7

Kofu
Katunuma-lKatunuma-2
Katunuma-3

47
32
40
38

5
5
6
5

Total

157

21

---------

---

Total

-[-

I

I

1
3
2
6

7.14

1
1
------_._-

10.00

2

2.86

12
6
6
3
-1-13.38

27
I

17.20

25

RESEARCHES CARRIED OUT IN 1959
Table 2.

Frequencies of deleterious and normal second chromosomes
in natural populations collected at three localities.

--------~=~=

Percentage of wild type flies
Population,--0--'
I

0-16.7---------:

Suyama9

Juriki
Kofu &
Katunuma

721iNo. of'16-.-8--25-.-0-2-5.-1--33.3-33 .4--4-1-.6 ! chromosomes--- --------

6
2

11

43

9

78

9

30

8

56

27

25

68

7

148

45

141

24

282

------

Total

37

35

I

13 .12_ _ _1_2_.41__ ~'_15_._96_~%

1

8_.5 _1______ __and semi-lethal genes partially dominant or produce local inbreeding insmall populations. The viability of each homozygous second chromosome
originated from natural populations was estimated by the ratio of Cy andwild type flies in the later generations. The classification of deleterious
and normal chromosomes is determined as follows; no wild type flies: lethal,
0.0-0.167 wild type flies:semi-lethal, 0.167-0.250:deleterious, 0.250-0.416:normal and more than 0.416:super vital second chromosome. The results
shown in Table 2 were obtained. The difference in number of the tested
chromosomes shown in Tables 1 and 2 was caused by the loss of somehomozygous strains in the later generations and the addition of a few
lethal and semi-lethal chromosomes.

8.

Effects of X-irradiation on biosynthesis of nucleic acidin Drosophila.
(By Toshifumi TAIRA and Toshiteru MORITA)

We have shown that the biosynthesis of nucleic acids in Drosophila ismarkedly inhibited by X-irradiation. The nucleotide, precursors of nucleic
acids, also are much influenced by irradiation. In the experiment in which
this was shown, 2rd instar larvae and early pupae (24 hrs. after pupation)
were subjected to 3,000 r irradiation. Twenty-four hrs. after irradiation,
each material was immediately homogenized with cold 10% aqueous perchloric acid. After centrifugation, the supernatants were neutralized with
5 N KOH in an ice-bath. The supernatants were used as the acid-soluble
nucleotide fraction and were charged on columns of Dowex-1-X 8-formate
resin. The eluting solutions ranged from 0.125N HCOOH to 4N HCOOH0.8M HCOONH 4 • Fractions of 5 ml were collected by the automatic fractioncollector. The absorbance of each fraction was read in the Beckman DU

RESEARCHES CARRIED OUT IN 195926

spectrophotometer at 260 m/-l. Paper chromatographical analysis, ratios of£260/£280, and quantity of inorganic phosphorus (measured in each main
fraction by means of a modified Fiske-Subba Row method) were used also
for the identification of nucleotides. The results are shown in Table 1.
Table 1.

Nucleotides

Effects of X-irradiation on nucleotide level in Drosophila(E260 /g in wet weight)
CMP+DPN

AMP

TPN

GMP

6.6
5.5

1.6
1.3

10.0
0.5

804

---

--~

I

ADP

GDP

ATP

12.6
4.0

-

1.1
1.6

-----

-- -

8.1
5.2

lOA
3.7

I
I

13.0
9.7

3.9
-----

1.2
10.6

i
I

-

10.2

-_.,

..

~

--------

Irradiated:
Larvae
Pupae

4.2

_

Control:
Larvae
Pupae

I

--

From these results, the following conclusions can be drawn: (1) Nucleotides of di- and tri-phosphate increase considerable in amount duringmetamorphosis. (2) Nucleotide levels are strikingly influenced by irradiation; nucleotide phosphorylations from monophosphate to triphosphate arestrongly inhibited.

9.

Distribution of the" Sex-Ratio" agents in the tissuesof adults of Drosophila Willistoni.
(By Bungo SAKAGUCHI and Donald F. POULSON*)It has been demonstrated by Malogolowkin and Poulson (1957, 1959) thatthe abnormal" sex-ratio" condition in D. willistoni is transmitted throughegg cytoplasm and that the agent in such eggs is capable of infectioustransfer into normal females converting them to the" sex-ratio" condition.
The" sex-ratio" agent has been experimentally examined from the view
point of its distribution among the various organs and tissues of the adultfly.
The "sex-ratio" strain SRB-3 was used in this experiment. This
strain was maintained for about twenty generation by back crossing tothe original "sex-ratio" strain with wild type males of Barbados-3 (B-3)
strain. _To test the possibility of transfer of the "sex-ratio" agent
from SRB-3 into normal B-3 abult females, blood, ovaries, wing muscles,
adipose tissues and gut were carefully taken out of two or three week old* Osborn Zoological Laboratory, Yale University, New Haven, Connecticut.

RESEARCHES CARRIED OUT IN 1959

27

SRB-3 females. The blood was directly sucked into a micropipette fromabdomens of the donor flies and immediatley injected into the abdomensof young virgin B-3 females. In the case of ovaries, muscles, gut, andfat body, the tissues were taken out of about fifty to one hundred donor
flies and repeatedly washed with Ringer solution.
These organs and tissues were then homogenized separately in a small
volume of Ringer solution using a small glass homogenizer. Small quantitiesof each homogenate were injected into the abdomens of the B-3 host flies.
The injected females were mated to males of their own strain. Bothcollection of eggs and examination of progenies were carried out in accordance with the procedure previously reported by Malogolowkin, et at(1959).

In the generation from B-3 females injected with blood or homogenatesof adipose, musclar, or ovarian tissue from the SRB-3 females, the progeny
in the early period of egg production consisted of approximately normal
proportions of females and males. However, as the injected flies aged the
proportions of females from every mother increased and finally, except insome cases, they produced mainly daughters. The results of second andthird generations showed almost complete unisexual bloods, again with a few
exceptions. In most of the cases of blood injection, the incubation period
from the time of injection to the beginning of the effect of the "sex-ratio"
agent was very short, only a few days, as compared to about twenty or
twenty five days after injection of homogenates of the other organs andtissues. It seems from these results that the" sex-ratio" agent is widelydistributed in the bodies of SRB-3 flies, viz. blood, fat body, flight muscles,
and ovary, and that there is a high concentration of "sex-ratio" agent in
the blood compared to other organs and tissues. In the case of injectionof gut there were no unisexual bloods among the progenies. However, it
is not clear at present whether the "sex-ratio" agent is present. in the
gut tissues, because the survival rate of injected flies was very low, only
2 out of 12 individuals.
A series of controls in B-3 flies were injected with blood or homogenatesof various organs and tissues of other B-3 flies was carried out. In theseexperiment one individual from the series of 12 ovarian injected and oneof the 8 muscle injected hosts produced the" sex-ratio" condition. This" sex-ratio" condition was maintained in further generations. It seems
that the "sex-ratio" agent may be maintained in a latent phase in individuals of the B-3 strain.
That the occasional males which survive in "sex-ratio" strains also
carry the "sex-ratio" agent was demonstrated by injection of blood into
normal B-3 females. In each case injected females soon produced only
daughters and the condition was transmitted in further generations.

28

RESEARCHES CARRIED OUT IN 1959

10.

Mass migrating activity in inbred lines derived from four
wild populations of Drosophila melanogaster.
(By Takashi NARISE, Kan-Ichi SAKAI and Shin-ya IYAMA)

Sixty five inbred lines derived from four wild populations of Drosophilamelanogaster, eleven strains from Tateba-sima (TB), fourteen from KatsuTable 1.

Analysis of variance of mass migrating activity of inbredlines of four wild populations of D. melanogaster.
TS and IG

KN and TB'_

Source of variation
----

d.f.

1

d.f.

mean square-------,------+-----~

PopulationLines win population
DensityDensity x population

1

23
5
5

Density x line win population I
Error-::-::---~-

mean square

115

232

9156.8639**
1960.9507**
6676.1363**
1769.4330**
305.7090
427.0929

7910.0822**
1063.7599**
7612.5266**
312.8952
413.6496*
215.8361

1
38
3
3
114

640

-. - - - - - - -c-:--=-----:-::-----* Significant at 5% level.

** Significant at 1% level.

irs 8

50%

IIG 28

/

~ 40

.....

/

.-i

'H

/

""
....
Q)

IG 14
- - T S 13

/

aJ30

tb

/

.~

TS 14

".".--j-

'H
0

/

",

-;:; 20
Q)

0

S
0.

................
........

~

..;

/

/

IG 6

..... 10
M

Q)

1
0
10

40

80

Initial number of flies in the original tube
Fig. 1.

120

RESEARCHES CARRIED OUT IN 1959

29

numa (KN), sixteen from Iguro-sima (IG) and twenty four from Te-sima(TS), were used in this investigation. After the completion of twenty
inbred generation in TB and KN and thirty in IG and TS, the massmigrating activity presumably caused by the pressure of population density
was investigated for each of the inbred strains. The details of the methodof investigation were given in the 1957 issue (No.7, pp. 72-73.) of thisAnnual Report. Counting of the number of flies migrated to the connected
tubes from the original one was made six hours after connection. The
analysis of data was made for KN and TB populations and for IG and TS
populations separately. Table 1 shows the analysis of variance of the massmigrating activity of the inbred lines from the four wild populations of
D. melanogaster. Migrating activity of a few strains of the IG and TS
populations is shown graphically in Fig. 1.
From Table 1 and Fig. 1 it can be concluded that the migrating activity
varies genetically not only among populations but also among inbred lines
within populations.
11.

Genetical aspects of two colour mutants of rats
caught in the suburbs of Misima(By Tosihide H. YOSIDA)

Two coat colour mutants of the rat (Rattus norvegicus) were caught inthe suburbs of Misima. One of them, a female rat, was characterized bya reddish yellow coat and pink eye. This rat was crossed with a CASTLE
black-strain rat with non- agouti coat (aa). All F , hybrids showed agouti
coat (A/a) and black eye. The F 2 progeny fell into four classes, namely
agouti and black eye (101 rats), non-agouti and black eye (35 rats), reddish
yellow and pink eye (49 rats) and light reddish yellow and pink eye (13The expected ratio ofrats), according to the dihybrid Mendelian ratio.
segregation in the above cases was 111.5:37.1:37.1:12.4. The above result
indicates that two independent genes, A for agouti coat and p for pinkeye, are involved in this mutant. The expression of A is conditional on
the presence of p. A and p together effect reddish yellow coat and pinkeye. When allele a occurs together woth p the coat is light reddish yellow. A pink eyed yellow mutant has been reported already by CASTLE(1915). The mutant found by the present author may be the same as thatobtained by CASTLE.
The other mutant rat (~) was characterized by brownish gray coat andruby eye. This mutant is similar to the Albany-strain which has lightbrownish gray coat and ruby eye. To find out whether the coat clour
gene concerned is the same as that of the Albany-strain, the two were

30

RESEARCHES CARRIED OUT IN 1959

crossed. All F , hybrids showed non-agouti coat and black eye, but the F%
segreg'lted into three types, namely non-agouti (29 rats), brownish gray(19 rats) and light brownish gray (11 rats). From the above investigation
it seems that the wild mutant and the Albany-rat have different coat colour
gene or genes, but have in common the agouti allele a.
By successive inbreeding of rats with new mutant characters, several
inbred strains, such as NIG-I, NIG-II, NIG-III, .... , are being established in the author's laboratory.

12.

Study on the new mutant" falter" found in the house mouse(By Tosihide H. YOSIDA)"Falter" (fa) is a recessive mutant wich appeared spontaneously in 1958
in a sib-mated stock (DBA/Ma-ce ) bred in the author's laboratory. In
homozygous condition, falter can be recognized by an abnormal side to side,
swaying walk.
The mutant was found in mice of about 10 days age.
The abnormality became more pronounced with advancing development,
and all the mice died at about 20 days.
By the mating of two heterozygotes (fa/ +), 81 offspring were obtained.
Among them 59 were normal, and 22 were abnormal, which indicates thatthe falter character is due to a recessive mutation. The expected segregation ratio is 60.75 and 20.25 (X 2 =0.202).
Table 1.

F 2 segregation with respect to genes p and fa
P fa

Types
Observ. No.
Expect. No.

23

7

27

9

p

Fa

p fa

Total

16

2

48
48

9

3
p=0.1-0.05

Characters similar to falter have been reported by FALCONER, 1951, (reelerr/), by DICKIE et al., 1949, (ducky du), and by HOECKER et al., 1954, (agitansag). These mutants allow development for a considerably longer periodof time and some animals grow to maturity.
Reeler (rl) is very similar
to falter and was proved to belong to the 3rd linkage group. In order
to investigate whether fa is in the same group or not, fa/ + mice (~)
were mated to NH-strain mice (-9-) which are characterized by having
genes p and s. Gene p (pink eye) is in the 1st linkage group and gene s(recessive spotting) is in the 3rd linkage group.
The F 2 progeny fromthe above mating segregated into four categories with respect to p andfa (Table 1). From the segregation ratios it seems that fa is independent

31

RESEARCHES CARRIED OUT IN 1959of the 1st linkage group.
Table 2.

F 2 sagregation with respect to genes s and fa--------- _ .. _ -

----

- -

Types

S Fa

S fa

s Fa

s fa

Total

Observ. No.
Expect. No.

30
27

5
9

9
9

4
3

48
48

----~._----

X2 =2.44

p=0.95-0.50

In relation to genes s and fa, the F 2 offspring segregated into four
categories as shown in Table 2. The segregation ratios indicate that fa is
independent of s, i.e. does not belong to the 3rd linkage group.

13.

Peripheral blood analyses of various
strains of mice and rats

(By Tadao HAMADA, Tosihide TABATA, Yoshinori KURITA
and Tosihide H. YOSIDA)

The present paper deals with the normal peripheral blood analyses ofvarious strains of mature mice and rats.
The methods used for the examination were as follows: 1) The blood
was withdrawn, always before meals between 9.00 a.m. and 10.00 a.m.,
Table 1.
Strain of mice
AjMs
AKRjlax
BALBjc]ax
C57BRjlax
C57jBLj6Ms
C57LjMs
C58jMs
CBAjMs
CFWjMsC3HjMsDBAjMaMs
dbajMs
ddjMs
DMjMs
Dl03jMs
NHjMs
S4jMs
SMjMs
SPSjMsSwiss Albino

Peripheral blood analyses of various strains of mice
R.B.C. (x10 6 )1,148.0

W.B.C. (x10 4 )
----

976.0
1.008.0
1,025.0888.0
892.0
1,085.0
934.0
893.0
887.0
1,200.0
993.0
885.0
870.0
963.0
1,109.0
1,078.0
1,001.0
865.0
964.0

5 mice were observed in each strain.

1.06
1.08
0.94
1.83
1.52
2.01
1.79
1.17
2.65
1.46
1.26
2.86
2.59
1.13
1.76
0.80
2.11
2.17
1.33
1.19

I
I

Neutrophils (%)---1-------43~--29.0
21.8
15.4
21.4
33.2
32.6
38.0
35.3
40.3
47.1
47.9
28.1
30.8
35.6
18.3
18.8
34.6
32.8
42.8

32

RESEARCHES CARRIED OUT IN 1959
Table 2.

Peripheral blood analyses of various strains of rat--------

Strain of rats

R.B.C. (x10')

WKA
W
Wayne
AlbanyFischer
BuffaloLong-Evans

812
777
827
858
821
887
759
788
917
855
890
880
806
875

W.B.C. (x 10')

Neutrophils (%)- - - - ~

N
SH
NIG
CW-I
CW-II
AN
WT

1.27
1.01
1.25
1.64
0.92
1.45
0.71
1.07
0.92
1.13
1.36
1.06
1.97
1.43

15
23
14
15
16
20
16
24
19
9.0
13
15
10

26

4-5 rats were observed in each strain.

from the right lateral vein in the middle part of the tail. 2) Hemocytometers for human hematology were used.
Blood counts for each animal
were repeated three to five times to minimize errors. 3) Smear preparations of the peripheral blood were made by the usual technique for leukocyte counts.
GIEMSA'S staining technique was used.
The morphologicalestimation of white blood cells was always followed by the usual hematologic technique.
The total number of cells observed per strain wereabout 10,000. The results of observations in mice and rats are shown in
Tables 1 and 2, respectively.
As shown in these tables, no marked differences were found between
the blood pictures of various strains. Noticeable is the fact that micesuch as A/HeMs, C3H/Ms, dba/Ms, etc., which show high incidence of
mammary tumor, have a comparably higher percentage of leukocytes,
while C57BL/6HeMs, BALB/cJax etc., strains with low tumor incidence,
have a higher percentage of lymphocytes.

14.

Reciprocal transPlantation of skin between rhino
and non-rhino mice
(By Shigekatsu TSUZI and Tosihide H. YOSIDA)

Rhino mice used in the present study were obtained in December, 1958,
from the Jackson Memorial Laboratory.
They are characterized by loss

RESEARCHES CARRIED OUT IN 1959

33

of hair and development of skin folds (HOWARD 1940). Experiments withskin transplantations between rhino and non-rhino mice have been carriedout by DAVID (1934) and FRASER (1946). According to DAVID (1934), hairlessskin grafts behaved autonomously on a normal host, while FRASER (1946)
demonstrated in young mice that the rhino skin on normal hosts showeddepilation in the center; on the borders, however, a zone of hair persisted.
The results obtained by the present authors from reciprocal transplantations of skin between adult rhino and non-rhino mice are as follows.
Rhino skin grafts on non-rhino hosts had on the surface the appearanceof reddish and smooth sloughs about one week after operation. Later,
the grafted skin became thicker and the characteristic skin folds appeared.
After a month, the grafts showed the characters of hyperkeratosis. It
is noteworthy that thin hairs appeared on the adult rhino skin which wasgrafted on non-rhino hosts 10 to 20 days after operation, but were shedcompletely about one month later.
In order to investigate why thin hairs developed on rhino skin grafts,
the following experiments were performed: (1) Rhino skins were graftedon other rhino hosts. (2) Rhino skins were retransplanted to the sameplace of the same host in various orientations. The results obtained fromthese experiments were similar to those obtained from those mentioned
above.
Thus, it seems that the temporary growth of thin hairs on thetransplanted rhino skin was due to a mechanical stimulation by the operation.
Non-rhino skin grafts on rhino hosts showed typical hair growth about
one month after operation.

15.

Study on phosphatase activity in rhino mouse skin
(By Kazuo MORIWAKI)

A mouse homozygous for the mutant gene rhino (hr'·") begins to lose its
hair at the age of about 14 days, while no such change occurs in a heterozygote. From experiments with reciprocal ectodermal transplants performed at an early stage, FRASER (1946) stated that after birth the hairloss character developed autonomously, freely from other control mechanisms
in the body (e.g. hormonal control).
As a biochemical property changed
with the appearance of the autonomous character, the author referred thechange to acid phosphatase activity of the skin.
It has been suggested
in this connection that in canaries the acid phosphatase has a relation tofeather formation through the fat metabolism of the skin (KOBAYASHI and
MARUYAMA, 1957). Also, in the mouse skin, the depilation seems to be accompanied by an accumulation of subcutaneous fat and activation of acid

RESEARCHES CARRIED OUT IN 1959

34

phosphatase. In mice younger than 2 weeks, both skins of rhino andnon-rhino animals showed normal hair growth and similar phosphatase
Table 1.

Comparison of acid phosphatase activity at various ages
in the skin of rhino homo- and heterozygotes.
Extinction of dephosphorylated PNP(-log T at 400 mll/7.5 mg wet tissue)Age

hr r "/+

Exp. no.
1.96
2.04
2.30
2.23
1.55
2.12
1.96

1
2

3
4
5
6
7

1.47
1.43
1.45

1.19
1.15
1.19

1.57
1.82
1.90
2.00
2.21

0.73
0.84
0.62
0.73
0.73

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

Average

2.02

1.45

1.18

1.90

0.73

activity. After 2 weeks, according to the progress of depilation, the enzyme activity in the skin of the homozygotes was shifted in comparison
with the heterozygotes (Table 1), and accumulation of subcutaneous fat
was observed in the former (TSUJI, unpublished).
A problem for further study involves which process induces the rhino
behavior, depilation, subcutaneous fat accumulation, or activation of skinacid phosphatase.
In this experiment, para-nitrophenolphosphate (PNP) was employed as
substrate of the enzyme, and the amount of dephosphorylated PNP after
incubation for 20 minutes at 37°C and PH 4.2 was estimated at 400 m/~.

16.

Effect of energy-rich phosphate esters on the swellingof mouse diaphragm(By Kazuo MORIW AKr)KREBS et al. (1949) found that the water content of a kidney slice is augmented by metabolic inhibitors such as KCN or 2,4-dinitrophenol, whichindicates that energy is necessary for keeping the water content in thetissues at a constant level. The present report deals with the correlation
between the swelling of mouse diaphragm and the decrease of the energyrich phosphate esters.

35

RESEARCHES CARRIED OUT IN 1959

- 3"C. air.
not ,hoked

o

10

20

Time in minutes
ofter immersion

Conditionsofincubation

o

10

20
10' P

30

1'/100 mg wet tissue

Fig. 1. Swelling and 10 ' P change in mouse diaphragm immediately
after removal under various conditions of incubation.

Immediately after excision, the mouse diaphragm contains the largest
amount of 10'P. The 10'P from such diaphragms was dissolved in 10%
cold trichloroacetic acid solution, precipitated by Ca-reagent and hydrolysedfor 10 minutes in 1 N HCI at 100°C.
The swelling of the excised diaphragm during 20 minutes of incubation
at 3°C in the KREBS-RINGER-phosphate solution was prevented by shaking
the tissue in the gas phase of oxygen or under air conditions at 37°C in
WARBURG vessels, whereby, to a considerable extent, the 10'P content wasmaintained. Under the same conditions, marked swelling of the diaphragm
was induced in the presence of 5 x 10- 5 M 2,4-dinitrophenol, whereby 10'P
content was reduced (Fig. 1).

~

I

20

I
I

rn

~
c

"g'

-- 3L:.o",r,

;'
:

'v~

37"~~~~~W2- 37'C,IO'WNaN"O,
37'C, N,I
I

snaked

:

10

0

.c
<)

I

~

c

8
~

a
a

20

80

ConditionsofTime in minutes

incubation

a

10
10' P
.r/l00 mg wet tissue

20

Fig. 2. lO'P content and recovery of the swollen diaphragm of mouseunder various conditions of incubation.

RESEARCHES CARRIED OUT IN 1959

36

The tissue swollen by incubation in the ordinary medium for 20 minutes
at 3°C returned to the initial state when incubations for 60 minutes in thepresence of 100% 02 or 20% (air) and shaking were applied, whereby theamount of 10'P reached a comparatively high level. The swelling describedabove failed to disappear when the tissue was treated with 5 X 10- 5 M 2,4dinitrophenol or 10- 3 M sodium azide or under anaerobic conditions, whereby
little of 10'P was found. At 3°C, the diaphragm was swollen for 20
minutes under air conditions, and the swelling continued for 60 minutes,
although a considerable amount of 10'P was found (Fig. 2).
In view of those findings, it seems likely that the extrusion of the water
penetraing into the tissue is conditioned by an endergonic mechanism
dependent upon the temperature.

17.

Interaction of Aegilops cytoplasm and various wheat
nuclear complements(By Hitoshi KIHARA)

KIHARA (1958) obtained a male-sterile strain of Triticum vulgare by substituting its nucleus into cytoplasm of Aegilops caudata.
In order to investigate the fertility effect of interaction between" caudata"
cytoplasm and various nuclear complements of Triticum, T. vulgare, T.
spelta, T. compactum, T. macha and T. durum were crossed twice to the
male-sterile strain (SBs) of T. vulgare. Pollen and seed-fertility of hybrids
in F , and B, generations of these crosses are summarized in Table 1.
F , and B I generations of SBs x T. vulgare (=SBg) and SBs x T. spelta wereboth male sterile but female fertile, giving fairly good seed-set by artificial pollination. It can be concluded from this result that these two
species have the same genotype with respect to the male sterility manifested in "caudata" cytoplasm.
Fls of SB8 x T. compactum were male-sterile but female-functional. In
the B I generation, male sterility segregation was observed. In some plants
male fertility was restored to a normal level. BI plants were too few
to predict the segregation ratio of male fertile vs. male sterile plants. Apparently, T. compactum carries a gene or genes responsible for restorationof male fertility. According to its effect the gene(s) must be recessive.
Crosses between SBs and T. macha produced only lethal Fls, indicatingthat T. vulgare and T. macha carry complementary dominant lethal genes
as noticed by other workers.
F,s of SB s x T. durum were male-sterile but female-fertile. A further
cross of this hybrid to T. durum produced plants (probably 2n=28) whose
male and female gametes were almost completely functionless. The result

37

RESEARCHES CARRIED OUT IN 1959

indicates that nuclear complement of T. durum cannot allow for a normal
development of male and female gametes when placed in "caudata" cytoplasm. This female sterility controlled by the nuclear complement of T.
durum seems to be recessive.
Table 1. Average pollen and seed fertilities of hybrids obtainedby crossing a male-sterile strain of Triticum vulgare
with five Triticum species.
-_. ---------

StrainPollenfertility(%)

Seed setting (%)- - - - -

Self

Cross
._---

SBg: Ae. caudatax(T. vulgare)S
SB 8 x T. vulgare=SB g
SB 8 X (T. vulgare)2 = SBlQ
SB a X T. spelta
SBs X (T. spelta)2
SB a X T. compactum
SB s X (T. com pactum )2
SBax T. macha*
SB 8 x T. durum
SB s X (T. durum)2**

0.3
0.2
0.0
0.0
39.9
4.9

0.0
0.0
0.0
0.0
1.3
4.7
51.6

23.2
41.2
56.3
27.9
39.1
33.4
53.6

2.0
0.8

39.2
4.1

* All F1s were lethal.
** Pistilloidy of anthers was observed.
18. Genome analysis in the genus Oryzal)(By Hitoshi KIHARA and Mitsuya NEZU)

We have collected a majority of the wild species of rice under a grantfrom the Rockefeller Foundation. At the start of our work on genome
analysis, O. sativa was crossed with 6 diploid and 3 tetraploid species, allbelonging to the Section Sativa ROSCHEVICZ, and seed fertility and chromosome pairing in F 1 hybrids were examined.
The F 1 hybrids between O. sativa and O. sativa var. spontanea or O. perennis showed 12 bivalents at meiosis but they were semi-sterile. The F 1 hybrids of the crosses O. sativa x O. glaberrima and O. sativa x O. breviligulata
failed to produce seeds, though chromosome pairing was very regular. In
the,:,meiosis of F 1 hybrids, O. sativa X O. officinalis and O. sativa x O. australiensis, very few bivalents were formed and no seeds were set. Fromthese findings it can be concluded that 4 diploid species, O. sativa var.
1)

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

w

00

Table l.

Chromosome pairing and fertility observed in the F, hybrids between 8 species of Oryza.
- -No. of cells I Bivalents
observed(Mode)

Combination~--------==-

Fertility (%)
Trivalents I Univalents---_ -

::0

Seed

,

Pollen>::0O. sativa x O. sativa var. spontanea (108xW0139)

30

12 (12)

0

0

37.0

(414xW0122)

35

12 (12)

0

0

63.9

(414 x W0032)

20

12 (12)

0

0

54.5

(414x W0149) .

55

12 (12)

0

0

57.4

O. sativa x O. glaberrima(414xW0040) I

45

12 (12)00

0.0

O. sativa x O. breviligulata(647 x W0042)

25

12 (12)00

0.0(W0025 x W0492)45

12 (12)

0

0

96.4

-

(647 x WOO08)62

0-1 (0)

0

22-24

0.0

0

"
O. sativa x O. perennis"

O. glaberrima x O. glaberrima
O. sativa x O. australiensis

tTl

(f)

tTl

-

..

(J

::r:
tTl

(f)

(J

>

::0
::0

M
tj
0

~
....,
>-<

Z

......

(!)

<:.n

(!)

O. sa#vax O. officinalis(563 x WOO06)

65

0-1 (0)

0

22-24

0.0

0

O. sativa x O. minuta (4x)(414xW0051) I

55

0-9 (6)

0-1

18-36

0.0

0-10

RESEARCHES CARRIED OUT IN 1959

39

spontanea, O. perennis, O. glaberrima and O. breviligulata, have homologous genomes while the other 2 diploid species, O. officinalis and O. australiensis, have different genomes from that of O. sativa.
So far as the crosses between O. sativa and the tetraploid species areconcerned, we could study only the F, hybrids of a cross between O. sativa
and O. minuta. They had at MI 18 to 36 univalents and 0 to 9 bivalentswith the mode at 6. Other workers have reported different results fromthe same cross. MORINAGA (1940) observed in most cases 361, though hefound occasionally 1 to 3 bivalents, while OKURA (1937) observed 1211+ 121 in
the majority of PMC's. NANDI (1936) studied hybrids between O. officinalis
and O. minuta and found that O. officinalis has the same genome as oneof the genomes of O. minuta.
Thus, it seems that O. minuta has one modified sativa and one officinalis
genome. This hypothesis will be tested by studying colchicine produced
amphidiploid between O. sativa and O. officinalis.
In addition, we are making diallel crosses between 18 species belonging
to all four sections, namely, Sativa, Granulata, Coarctata and Rhynchoryza(T. KATAYAMA & M. NEzu). On the basis of the F, hybrids from thesecrosses, we will analyse the genome constitutions of all four sections of
the genus Oryza.
19.

Application of SUMP method in taxonomic studies zn Oryzal)(By Hitoshi KIHARA and Tadao KATAYAMA)The surface structure of the lemma is an important taxonomic characterin Oryza, as was recognized by ROSCHEVICZ. In order to examine thedetails, we studied the lemma of 18 Oryza species by the convenient SUMP
(Suzuki's Universal Microprinting) method with the following results:(1) Section Sativa ROSCHEV.
The cells are square-shaped or rectangular. Their arrangement is regular. Each cell bears a round tubercle and has a smooth or rough surface. The tubercle gives the impression of a bursting membrane. Hairs
occur in most of the species.
(2) Section Granulata ROSCHEV.
The cells are rectangular and have irregular contours. Each cell has
a tubercle composed of several tuberclets.
(3) Section Coarctata ROSCHEV.
In general, the cells are slender and without tubercles. They are arranged in long stripes with deep grooves between them. Species belonging
11

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

40

RESEARCHES CARRIED OUT IN 1959

to this section show a great variation in structure. O. coarctata has stomata on the lemma.
(4) Section Rhynchoryza ROSCHEV.
The cells are polygonal, varying in size and are arranged like the scalesof a tortoise shell. They have in the middle a globe-shaped tubercle, fromwhich furrows radiate.

x40

x164

Fig. 1. Surface structures of the lemma in genus Oryza.
A: O. sativa, B: O. granulata, C: O. coarctata, D: O. subulata

20.

Studies on germination in Oryza1)(By Tadao KATAYAMA)

Germination behavior
The germination behavior, i.e., germination percentage and rate, werestudied using 18 species of genus Oryza collected abroad. Forty to 200
grains of each strain were freed from the glumes and sown in a petri-dishkept at 30°C. Experiments were made 8 times between April 10 andJune 28.
The species can be classified into four groups with respect to their germination behavior according to the data obtained. First group: Germina·
1.

l!

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

RESEARCHES CARRIED OUT IN 1959

41

tion occurred within 1-2 days after sowing: Afterwards no germination
occurred. O. sativa and O. glaberrima belong to this group. Second
group: Germination started within 2-3 days after sowing and afterwards
its percentage gradually increased. Even 270 days after sowing someseeds still germinated. O. sativa var. spontanea and O. perennis belong
to this group. Third group: Germination started within 5-6 days aftersowing. During the following 3 weeks, the germination rate increased,reaching the maximum at about 70%. O. granulata belonged to this group.
Fourth group: Germination started within 2-3 days after sowing. The
germination percentage gradually increased and reached the maximum
10 days after sowing. O. alta, O. australiensis, O. brachyantha, O. bre·viligulata, O. eichingeri, O. latifolia, O. minuta, O. officinalis, O. ridleyi
and O. subulata belong to this group.
II. Germination test with plant hormones
Forty seeds of each of 13 Oryza species were soaked 12 hours in water at
30°C and treated with Indole acetic acid (IAA) and gibberelline (GIB) in
a concentration of 1, 10 or 100 ppm for 12 or 24 hours in order to examine
the physiological effects of plant hormones on the germination behavior of
Oryza species.

100

1

2

.... ....

3

4

10

10

-

10

10

20

days

Vertical axis; per cent germination.
Horizontal axis; number of days after sowing.
Fig. 1. Germination behavior in the genus Oryza.

Effects of the treatments varied with the species. Germination of thecultivated rice was little influenced by hormone treatment. Germination
rate of wild species treated for 12 hours showed maximum acceleration by
100 ppm GIB. Only a few species (0. australiensis, O. granulata, O. lati·
folia), responded to IAA of the same concentration. When applied for 24hours, IAA and GIB showed almost the same effect. The effect wasstronger than that of the 12 hour treatment in all species except O. perennis which responded in the same way to both 12 and 24 hours treatments.
No effect on breaking dormancy of wild rice was detected.

RESEARCHES CARRIED OUT IN 1959

42

21.

Studies on the flowering time of genus Oryzal)(By Tadao KATAYAMA)

A number of strains of cultivated and wild rice collected abroad belonging to 18 species of the genus Oryza, were grown in the greenhouse. Theirflowering and its duration were investigated. Observations were made 21
times between September 12 and November 18 for several strains of eachspecies. The flowering time means the time of opening of the lemma andpalea of the spikelet that flowers first on a panicle. The duration of
flowering time indicates the interval between opening and closing of aflower.
Results of the observations on the flowering time are given in Fig. 1.
O. sativa var. spontanea and O. perennis were not much different from
cultivated rice which flowers in the morning. All the other wild species
opened earlier in the morning than the cultivated rice except O. australiensis
Fig. 1.

Species

4
I

I

The flowering behavior of Oryza species.
Flowering time (4-17 o'clock)'Flowering' Flower_ _ _ _ _ _~responseing
5 6 7 8 9 10 11 12 13 14 15 16 17 'to cli~.atic dl,lracondItIOns tlOn
I
I
I
I
I
I
I
I
I
I
I
I
I
---------------------_.-

O. sativa
O. s. v. spontanea
O. perennis
O. glaberrima
O. breviligulata
O. stapfii
O. ojficinalis
O. australiensis
O. minuta
O. malabarensis
O. latifolia
O. altaO. eichingeri
O. granulata
O. brachyantha
O. coarctata
O. ridleyi
O. subulata-----~

1)

I

L
L
L
L
L

M

T
T
T
T
C
C
C
L

L

M
M
M
L
M
L
L
L
L
M
L

C

S

C
T
T
T

"S
L
L

- - - - - -This work supported by Grant RF 57080 from the Rockefeller Foundation.

RESEARCHES CARRIED OUT IN 1959

43

and O. coarctata, both of which opened in the afternoon.
Temperature, relative humidity and the time of sunrise were correlatedwith the flowering time of each species. The species studied can be classified into three groups with respect to their flowering response to climaticconditions. In the first group, indicated with" C" in Fig. 1, the flowering
time was very stable regardless of the climatic conditions. In the second
group, indicated with "L" in Fig. 1, the flowering time varied mainlywith the time of sunrise, i.e., it became gradually later from September
to November in accordance with the calendar. Very few wild species
belong to this group. In the third group, indicated with" T ", the flowering time was mainly determined by temperature.
As the relative humidity during the experiment was kept higher than
70%, the critical humidity at which flowering does not occur could not be
determined.
The species studied were classified into three groups based on flowering
duration; (1) species indicated by M showed approximately the same duration of flowering as that of O. sativa, (2) species indicated by L showed
relatively longer duration than O. sativa, and (3) species indicated by S
showed a shorter duration than O. sativa. As shown in Fig. 1, species
distantly related to cultivated rice showed in general not the same but
either longer or shorter duration.
In O. ridleyi the male-sterile spikelet at the top of each rachis always
opened earlier than the male-fertile spikelets.

22.

Photoperiodic responses of Oryza species I l l i(By Tadao KATAYAMA)(11 The materials used in this experiment include two cultivated species,
O. sativa and O. glaberrima, and wild growing O. sativa var. spontanea
and O. perennis. Several strains which have the" critical day-length" ofabout 13 hours were selected for this experiment. They were grown inan experimental short-day field and in pots.
Studies in this experiment, were the relationships between the photoperiodic reaction and age of plants on one hand and between the former
and number of short-day treatments on the other hand.
Up to the critical short-day length, the number of short-day treatments
necessary to induce flowering increases proportionally with the length ofillumination (Table 1). With the same short-day treatments flowering was
accelerated in relation to the increased number of treatments. Photoperiodic
sensitivity increases with the age of seedlings, reaching to the most sensitive
11

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

44

RESEARCHES CARRIED OUT IN 1959
Table 1.

Photoperiodic sensitivity of some rice strains in thevicinity of critical day-length.
Age inStrains

12:35*

I

13:05

12:50

_______________ i _ _,_,days

6**9 15 21 27

50
35
50
50
50
50
50
50
50
50
50

--------

6 9 15 21 27

6+ + + + ++ + ++ + + + ++ ++ + + , +++ + + +

+ + +

+ +

+ +
+ + +
+ +
+

+
+ +
+

+

+ +

+ +

9 15 21 27

I

Kyoto Asahi I
fI

fI

108
124
230
W0026
W0039
W0106
W0107
W0120
W0149

II

-------* Day-length of short-day treatment** Number of treatments+ Effective- Ineffective

age when the plants can respond to the" critical day-length". The numberof treatments required for induction of flowering was smaller in plants
collected from higher latitudes than in those collected from lower latitudes.
Furthermore, it seems that this number increases in the order, O. sativa,
O. glaberrima, O. sativa var. spontanea and O. perennis.
(2) Variation of photoperiodic sensitivity and critical day-length werestudied in each species. The variations were found to decrease in theorder, O. sativa, O. glaberrima, O. sativa var. spontanea and O. perennis.
As a whole, cultivated rice showed a greater variation than wild rice.
(3) The author previously reported (Katayama et al. 1959) a positive
correlation between the critical day-length and the latitude of the habitatof the wild species. The same experiment was carried out with 383 strainsof cultivated rice, collected from 10 countries in various latitudes. The
data indicate that there is a similar correlation between the critical daylength and latitude of the habitat. However, the correlation was lesspronounced in the cultivated rice than in the wild rice.

RESEARCHES CARRIED OUT IN 1959

23.

45

Monosomic analysis of lethal factors in common wheat(By Koichiro TSUNEWAKI)

Identification of chromosomes carrying complementary lethal factors
widely distributed in common wheat varieties is the subject of this investigation.
The F 1 hybrids from crosses between Prelude and Kharkov or Jones
Fife were all semi-lethal, bearing about 10 seeds per plant. In the F 2
generation the cross Prelude x Kharkov produced a ratio of 41 lethals to
30 normals and the cross Prelude x Jones Fife, 57 lethals to 43 normals.
Both closely fit a 9:7 ratio indicating that lethality is caused by two
dominant complementary genes. Kharkovand Jones Fife apparently carry
the same gene because the cross (KharkovxJones Fife) FlxPrelude produced 252 lethal plants and a single normal plant. The normal plant might
have been produced by out-crossing.
In order to determine the location of the complementary lethal genes,
the monosomic series of Prelude was crossed with Kharkov and the Kharkov
monosomic series with Prelude. Monosomic series of Prelude and Kharkov
were derived by McGinnis and Jenkins, respectively, from the Chinese
Spring series. Both series, particularly that of Prelude, are in an earlystage of development. Because the monosomic plants used for crosses
were obtained by self-pollination, it is expected that genes on a disomic
chromosome can be heterozygous or homozygous for either dominant orrecessive factors. However, genes on the monosomic chromosome must
be derived from Prelude in the Prelude monosomics and from Kharkov inthe Kharkov monosomics.
In the non-critical monosomic lines, one can expect an Fl ratio of either
1:0, 1:1 or 0:1 for the semi-lethal vs. normal plants depending upon the
genotype of the -9- parent. On the other hand, only a 1:3 ratio is expected
for the critical monosomic line, because all disomic F1's must be semi-lethal
and the monosomics, normal. Based on these considerations, the actual F 1
ratio obtained for each monosomic line was compared with the nearest ratio
among the three expected for the non-critical line and the 1: 3 ratio expected
for the critical line.
All F 1 lines from crosses between Prelude monosomics and Kharkov,
except those derived from mono-V, fitted one of the three ratios possible
for a non-critical line. The ratios obtained from lines of mono-V fitted
the critical 1:3 ratio only, indicating that chromosome V of Prelude carries
one of the complementary genes. In the crosses between the Kharkov
monosomics and Prelude, all F 1 lines except those derived from mono-XIII
satisfied one of the three ratios expected for a non-critical line but disagreed

46

RESEARCHES CARRIED OUT IN 1959

with ratio for the critical line. On the other hand, the Fi ratio obtainedin the progenies of mono-XIII failed to fit any ratio for a non-critical line
but satisfied the ratio expected for the critical line. These results indicatethat chromosome XIII of Kharkov carries the other gene for lethality.
The Prelude gene on chromosome V will be designated as Lei and the
Kharkov and Jones Fife gene on chromosome XIII as Le2. The Lei geneis identical with Caldwell and Compton's Le gene. It is yet uncertain
whether their Le2 gene is the same as the gene of Kharkov and Jones Fife.
24.

Developmental studies in the genus Oryza I.
The process of embryo sac formation. lI(By Yukio DOIDA)

From the view point of comparative morphology, the process of embryosac formation was studied in some species of the genus Oryza. The
materials used are listed in Table 1.
Table 1.
SpeciesMaterials and Origin
Strain No.

I

Origin- - - - - - - - - 1 - - - - - - - - - ------

O. sativa
O. glaberrima

O. breviligulata

W0017
W0009

Nat. Inst. Genet.
Central Rice Res. Inst.,
Cuttack, India. (C.R.R.I.)
C.R.R.I.
I C.R.R.I.

O. ridleyi

W0001

1

O. alta

Kyoto Asahi
W0026

Ba~gkok_O. sativa L.
" Kyoto Asahi", a rice of Japonica type, was used. Its embryo sac
formation was found to resemble the so-called" Polygonum type" whichis the commonest type in the angiosperm. An archesporial cell (=embryosac mother cell) undergoes normal meiosis resulting in a tetred. Threecells at the micropylar end degenerate. The innermost cell undergoes threesuccessive mitoses, and eight nuclei are formed. The three nuclei at the
micropylar end form the egg apparatus and the three at the charazal end
form the antipodal cells, the remaining two constituting the polar nucleus.
Thereafter, the three antipodal cells become multinuclear or polyploid.
O. glaberrima Steud.
The process of embryo sac formation is almost the same as in common
1)This work was supported by Grant RF 57080 from the Rockefeller Foundation.

RESEARCHES CARRIED OUT IN 195947

rice. The three antipodal cells hypertrophy and become multi-nuclear or
polyploid.
O. alta Swallen
The process of embryo sac formation is almost the same as in common
rice. The antipodal cells become multi-nuclear or polyploid. Twenty-fourbivalents were observed at MI in PMCs.
O. breviligulata A. Cheval et Roehr.
The process is the same as mentioned above.
O. ridleyi Hook.
The three antipodal cells show a secondary development and the antipodal
apparatus consists of several cells, sometimes ten or more. Thus, theprocess in O. ridleyi is somewhat different from that described above forthe other 4 species.
In the 5 examined species, the megasporangium consists of a nucellus
and two integuments which envelop the mature embryo sac. The ovule
is anatropous. The nucellus is well developed at the chalazal end.

25.

The karyotype and intra-genome pairing of chromosomesin haploid plants of Oryza glaberrima(By Chao-Hwa Hu and Y6 TAKENAKA)

Oryza glaberrima Steud., cultivated in the West Africa, is considered to
be a distinct species since it differs in several characters from the common
cultivated rice, O. sativa, and the F 1 hybrid with O. sativa is highly
sterile. Haploid plants of O. glaberrima were obtained from a twin seedling. The chromosomes in root tip cells and PMCs were observed, andthe results were compared with those previously obtained in haploid plantsof O. sativa.
In both O. glaberrima and O. sativa nine types of chromosomes could
be identified on the basis of size, position of constriction, and presence orabsence of satellites and secondary constrictions. No karyotypic difference
could be found between the two species. Also in the frequencies of bi- andtri-valent chromosomes and of secondary associations in PMCs, the two
species showed no significant difference. It is known that the diploid F 1
plants of these two species show no disturbance in chromosomes pairing.
It was concluded, accordingly, that these cultivated species of Oryza might
have cytologically the same chromosomes.

RESEARCHES CARRIED OUT IN 1959

48

26.

Systematic studies in the genus Oryza by statistical methods!)(By Hiroko MORISHIMA and Hiko-Ichi OKA)

For evaluating interspecific relationships in the genus Oryza, the writers
have been engaged in statistical-taxonomical studies of the strains kept in
the collection of the Institute. The results of a preliminary investigation
were reported last year (Annual Report No.9: 35-36); this paper presents
the results of further studies by two methods, factor analysis and Sokal's
weighted variable group method, of between-species correlation matrices.
Between-species correlations based on measurements of 42 characters,
which were assumed to be a quantitative index of the degree of resemblance between species, were calculated among 16 species.
Of the 42 characters, 22 were recorded qualitatively using code numbers,
and the rest were measured in standard deviation units. The correlation
matrix obtained from the data was analysed first by the averoid methodof factor analysis, extracting the first to fourth factors.
Then, the 16
species were scattered in the space defined by the factor axes. The scatter diagrams showed that certain species are gathered in clusters.
Secondly, the same correlation matrix was analysed by Sokal's method
to illustrate interspecific relationships in a tree-like diagram (Fig. 1). The
results obtained by both methods were comparable and were in generalin accord with the classification of the genus by Roschevicz (1931).
However, Roschevicz's Section Sativae can be sub-divided into two distinct.--

O. aubulata (18)
O. bracbyaDtba (15)
O. ridle:r1 (14)O. granulata (13),--

O. minuta (9)O. otti.inalia (10)O. malabarenaia (12)O. alta (8)O. latitclia (7)O. ei.bingeri (11)O. auatralien.ia (8)O. breYiligulata (5)O. glaberrim& (4)O. oatiYa '(1)O. perennia (3)O. aati .•pontane. (2)

-.4

-.2

0

.2

.4

.8

.8,

1.0

Fig. 1. Tree-like diagram showing interspecific relationships obtainedby Sokal's weighted variable group method.
1)

This work was supported by Grant RF57080 from the Rockefeller Foundation.

RESEARCHES CARRIED OUT IN 1959

49

groups of species, one represented by O. sativa, O. perennis, O. sativa f.
spontanea, O. glaberrima and O. breviligulata (called the" Sativa Group "),and the other by O. officinalis and the other six related species (called the"Officinalis Group "). Section Coarctatae (0. ridleyi and O. brachyantha) ,as well as Section Rhynchoryza (0. subulata), are found to form distantgroups. It is suggested from the results that the main lines of evolutionary progress in the genus Oryza might be directed to the "Sativa" and" Officinalis " groups, leaving other species outside the main lines.
(Published in Evolution 14:153-165, 1960.)

27. Varzation studies in wild rice species, Oryza pernnis
and O. sativa f. spontanea, by multivariate analysis ll(By Hiroko MORISHIMA and Hiko-Ichi aKA)

The two wild rice species, O. perennis and O. sativa f. spontanea, caneasily be crossed with each other and with the cultivated rice, O. sativa,
to produce fertile hybrids.
They have been, however, considered to be
distinct species. The major difference so far recognized between them isthat the former is perennial and has rhizomes, while the latter is annual.
In order to look into the pattern of variations in these wild rice species,
a number of strains belonging to either of the two, collected from India,
Thailand and Malaya, were grown in Misima, and records were taken forvarious characters. In all characters, the strains showed a continuous rangeof variation.
The multi-character variation in this group was then investigated by the"component analysis" method. The original data were taken for 60 strains,among which seemingly perennis, intennediate and spontanea types were
included. Correlation coefficients among five characters, i.e., anther length(A), length/width ratio of grains (G), ligule length (L), panicle length (P),and branch number per panicle (B), were caluculated from the data.
Then, two principal components were extracted from the resultant correlation matrix. The two component axes gave the following combinationsof characters.
Xl = 1.00 A+0.94 G+0.26 L+0.44 P-0.52 B .
X2=0.12 A-0.28 G+1.00 L+0.74 P+0.86 B .
The 60 strains investigated were then scattered on a plane defined by
the Xl and X2 axes (Fig. 1). The scatter diagram showed no particularcluster of strains.
We cannot know, however, from the results of this computation what
1)

This work was supported by Grant RF57080 from the Rockefeller Foundation.

RESEARCHES CARRIED OUT IN 1959

50

% of regeneration:•.. •.. •

0.8

0.6

•

0 - 20 %II-

20 - 80 %- - 80 - 100%

.....

•
0.4

•
0.2

•

• •
1.2

Fig. 1.

••

1.4

1.6

1.8

2.0

2.2

2.4

Xl
Strains of Oryza perennis and O. sativa f. spontanea scattered in theplane defined by the first and second component axes.

the component axes mean.
We computed two discriminant functions in
an attempt to find their meanings, one (Y l ) to maximize the difference
between seemingly perennis and spontanea types, and the other (Y2) tomaximize the difference between wild and cultivated strains. They were
found to be
Yl = 1.00 A+0.64 G-O.02 P+0.46 L ,
Y 2 =1.00 5+0.38 D~0.20 W-O.13 N ,andwhere A, G, P and L are the notations used in Xl and X2, and 5, D, W
and N represent measurements for degree of shedding, degree of seed
dormancy, grain weight, and grain number per panicle, respectively.
Having then examined correlations between the 'scores given by Xl, X"
Yl and Y2, we found high correlation coefficients between Xl and Yl(r=0.906), as well as between X, and Y 2 (r=0.519). It may then be assumed that the first component Xl shows differentiation in the directionof perennis vs. spontanea types, and the second component X 2 shows thatin the direction of cultivated types.
A further attempt was made to investigate the pattern of variation inthis group. Using data for 12 populations belonging to either of the two,t

RESEARCHES CARRIED OUT IN 1959

51

species grown in Taichung, Taiwan, a discriminant function to maximize
the variance among populations relative to that within populations wasconstructed. It was
Z=-0.09Aw+1.00L+0.64P-0.67 W. (Aw: Awn length)
The scores given by this formula seemed to discriminate clearly the perennis and spontanea types. Frequency distributions of the score, in each
population and among populations, were then examined. Among populations, there appeared a continuous array of intergardes from the perennis
to the spontanea types. The variation within populations, which might beindicative of heterogeneity in the population, was narrow in range in
spontanea types, and was wider in intermediate and perc)znis types.
The percentage of regeneration after cutting at maturity was examined,further, with the view to measure the perennial vs. annual habit. Populations of the perennis type showed higher percentage than those of the
spontanea type, but the two species could not be clearly discriminated by
the percentage, as shown in Fig. 1. From these variation studies, it was
concluded that in so far as Asian materials are concerned, there is noclear distinction between O. perennis and O. sativa f. spontanea, while thetwo species may be considered to represent both extremities of a continuous variation in the group to which they belong.

28.

Hybrid sterility relations among cultivated and wild strains of ricel)(By Kokichi HINATA and Hiko-Ichi OKA)

In his previous paper on phylogenetic differentiation of Asian cultivated rice
varieties, the junior author (OKA 1953) reported on hybrid sterility relationships among the varieties. Hybrid sterility was scored by means of "reaction types" indicated by a combination of plus and minus signs; a plus
(or minus) sign respresents the pollen fertility of the F, of a given strain
crossed with a certain test strain being higher (or lower) than 87.5 %.
Thus, the percentages of good pollen, though measured in each crosscombination, could not be fully utilized to deduce a final conclusion by
this method. In order to overcome this defect, we have attempted in thisstudy to use "component analysis" and other mathematical techniques fortreating the data.
A number of wild rice strains, collected from various localities of India
and belonging to Oryza perennis or O. sativa f. spontanea, were used as
the materials for crosses. They were each crossed with five test strainsof O. sativa, 108, 414, 521, 563 and 647. The first two, 108 and 414, be11

This work was supported by Grant RF57080 from the Rockefeller Foundation.

52

RESEARCHES CARRIED OUT IN 1959

long to the so-called "Continental" or "Indica", 521 and 563 to the" Temperate-Insular" or "Japonica" and the last one, 647, to the "TropicalInsular" type.
These are the same strains used by the junior author(OKA 1953) before; therefore, his old data for many cultivated strains could
be analysed together with the data for the wild strains.
Y2
250

6 x x

6
200

..
-15d36 V.,........
6 x ~~ x x.,~

M
00 0

oo 0

c9

j

o

150 06 iI Ia
Ib

0

0"

• I lIa O.satlVQ.
... rIa (OKa 1953)• • lIb~ • ••

OOnoOD
OOu-.o.

• •

••
•
•

x • 0. pel"Bnnis and O. sativa f. s ponravea
100'---'--'-------'---------"-0.
100'
200 Y,
Fig. 1. Distribution of wild and cultivated strains in the plane
defined by the first and second component axes extracted fromthe data for hybrid sterility.

In the original data, each strain to be examined was represented by aset of five good-pollen-percentages (A, B, C, D, E), one each from the fivetest strains. Taking those values as variates of the strain, the first andsecond components were extracted from the data for cultivated strains.
The two components gave weights (relative magnitudes of direction cosines) to the five variates as follows:

Y 1 =-0.97 A-0.56 B+1.00 C+0.93 D+0.S6 E,
Y z = 0.57 A+0.77 B+0.02 C+0.16 D+1.00 E .
Secondly, from the same data, a discriminant function was computed to
combine the five variates so as to maximize the difference between the" Continental" and the "Insular" types, into which the strains had been
divided. The discriminant formula was found to be
Y 3 =-1.00 A-O.IS B+0.12 C+0.58 D+0.62 E .
Comparing the Y 1 and Ys formulas, we find that they have the samearrangement of signs for the five variates.
Further the scores given by another discriminant function, computed byOka (1956) to classify the "Continental" and the "Insular" types by combining measurements for various characters, showed a high correlation

RESEARCHES CARRIED OUT IN 1959

53

coefficient (r=0.65) with the scores given by Y 1 • It may then be assumed
that the first component, Y 1 , represents the differentiation of these two
phylogenetic groups in O. sativa.
Then, we examined the scatter diagram in Fig. 1, which shows the distribution of the strains used in the plane defined by the two component
axes. The" reaction-types" classified by the old method are also shownin the figure. It is recognized in the figure that the old and new methods
have given comparable results, though the continuity of variation could
be illustrated only by the new method. Further, an interesting fact foundin Fig. 1 is that most wild strains appear to be intermediate between the" Continental" and "Insular" types in the score given by Y 1 , while they
have larger scores for Y z than the cultivated ones and are located in theupper part of the diagram. It may then be assumed that the second
component, Y z, represents the direction of variation from wild to cultivated types.
In the original data, a greater part of the wild strains tested showed
high fertility with all of the five test strains, though the latter showsterility in their mutual F1s. It may then be said that the differentiation
among wild strains toward the "Continental" and "Insular" or the "Indica" and "Japonica" types is not much advanced.
However, in thewhole group including both wild and cultivated forms, the major directionof differentiation might be toward the "Continental" and "Insular" types.
After subtracting this component, the remaining part of the variation
given by the second component, Y2, might represent the differentiation ofwild and cultivated types. Thus, the scatter diagram given by the twocomponent axes may be regarded as a pictorial representation of thedevelopment of cultivated types from wild ones.

29.

Variation in hybrid sterility with certain test strains within
populations of wild rice 11(By Hiko-Ichi OKA and Wen-Tsai CHANG)

Populations of wild rice (0. sativa f. spontanea and O. perennis), collected from various spots in India, were raised in the experimental fieldin Taichung, Taiwan, and a number of plants taken at random werecrossed with two test strains of cultivated rice (0. sativa).
Thus, twokinds of F 1 and self-pollinated seeds were obtained from each plant. Peiku(or 108, Indica) and Taichung No. 65 (or 504, Japonica), whose F 1 shows semi1)

This work was supported by Grant RF57080 from the Rockefeller Foundation.

54

RESEARCHES CARRIED OUT IN 1959;-sterility, pollen fertility being about 45 %, were chosen as test strains.
Variations in pollen fertility found among the F 1s and self-pollinatedlines thus obtained are given in Table 1. As the data in the table show,
in the case of a population of O. sativa f. spontanea, W203, all self-pollinated lines as well as those crossed with 108 showed high fertility, andsemi-sterility was found only in a few lines crossed with 504. As reported in another paper (HINATA and OKA), the F 1s between this species andvarious cultivated rice varieties are generally fertile, though between thecultivated ones, those distantly related show semi-sterility in their F,s.
The above-mentioned fact suggests, however, that the populations of
O. sativa f. spontanea might contain plants which produce semi-sterile F,swith a certain cultivated type.
III the case of the W208 population of O. perennis, semi-sterility occurred not only in the F,s but also in self-pollinated plants. The mechanismof sterility in self-pollinated plants and in the original population is a problem to be studied.
Table 1. Variations in pollen fertility among self-pollinated lines of wild
rice and their F1s with two test strains of O. sativa.
Population
Self(spontanea) 108 x
504 x
Self
W208
(perennis) 108 x
504 x
W203

100 95
10 8
4 4
8
2
1
3

18
1

8
11
11
7

2

531
122
2

2

1

10

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

30.

An investigation of floating habit in u ild rice species 11(By Hiroko MORISHIMA, Kokichi HINATA and Hiko-Ichi aKA)

In tropical Asian countries, delta areas of rivers are flooded in the rainyseason and the depth of water reaches several meters. In such areas, floating rice is the only food crop which can be grown. The floating habit ofrice is potential; when deep water conditions are given, rapid elongationof internodes, branching of culms, and development of adventitious roots
from water-submerged nodes take place. However, if grown in an ordinary paddy field, the floating varieties show the same growing habit as
the non-floating ones.
1)

This work was supported by Grant RF57080 from the Rockefeller Foundation

RESEARCHES CARRIED OUT IN 1959

55

Oryza perennis and O. sativa f. spontanea are wild species closely related
to O. sativa. The former usually grows in deep-water swamps and seems
to be of floating nature. In order to examine if they have floating habit
or not, 10 strains, belonging to O. perennis and O. sativa f. spontanea and9 cultivated strains, collected from various localities in India and Thailand,
were tested in a pond as follows: Seedlings were raised in a nursery bed
for two months and were transplanted at the bottom of a pond 72 cm
deep. Starting from the tenth day after transplanting, water was added
to the pond increasing its depth by 4 cm a day. The plants then remainedin the pond for two months.
Records were taken regarding (1) numberof internodes longer than 3 cm, (2) total length of elongated internodes,(3) number of branches per tiller, (4) number of nodes with adventitious
roots, (5) presence or absence of rhizomes, and (6) tiller number. It was
assumed that floating ability would be measured by characters 1 to 4.
The results of this experiment showed that all strains seemingly belonging to O. perennis had floating ability, and those belonging to O. sativa
f. spontanea had it also to some extent. Among cultivated strains, however, those with and without floating habit could be distinguished relatively clearly. Rhizomes were found in two strains of O. perennis.
It was then suggested that O. perennis as well as O. sativa f. spontanea
might generally have floating ability, though its degree may vary among
populations in accordance with the depth of water in their habitat. As
reported in another paper (MORISHIMA and OKA), these two wild species
show a continuous array of intergrades and cannot be distinctly classified.
It is possible that the differentiation of perennis and spontanea types
results from adaptation to different depths of water.
31.

Variation in seed dormancy of wild rice species!)(By Hiroko MORISHIMA and Hiko-Ichi OKA)

It seems that dormancy of seeds is an important character distinguishing the wild from the cultivated types of rice. Twenty eight strains of
Oryza perennis and O. sativa f. spontanea, collected from various localities
in India, and 92 cultivated strains (0. sativa) from various Asian countries,
were tested for seed dormancy. Seeds harvested about 20 days after
heading were kept in a desiccator, and germination tests with hulled andunhulled grains of each strain were made three times, 50, 90 and 180 days
after heading. The germinating capacity and the average number of days
required for germination were thus obtained for each sample. In order1)This work was supported by Grant RF57080 from the Rockefeller Foundation.

RESEARCHES CARRIED OUT IN 1959

56

to evaluate the germinating activity of a sample as a whole, however,
the two measurements were combined as follows:"Germma. t·mg actiVIty.. " = (%f germmatlOn. . .mays
10d) x ll-No.ofdays
00 0
10 -.
The number of days required for the "germinating activity" to reach
40 % was then calculated for each strain as the measure for the degreeof dormancy. Frequency distributions of the number of days thus obtainedwith hulled seeds are given in Table 1.
Table 1.

Variation in number of days required for "germinating
activity" reaching 40 %. (Tests with hulled seeds)
No. of days

Type
40

PrennisI ntermediate
Spontanea

60

80

100

120

140

160
2

5
2

2

180 ormore

No. ofstrains

2

4

3

10

6

11

Cultivated(0. sativa)

86

3

1

92

As shown in the table, wild strains generally had stronger dormancy
than the cultivated ones. It seems, however, that some strains of intermediate type between perennis and spontanea had a relatively weak
dormancy like that of cultivated strains. If the hulls were not removed,
the seeds of wild strains showed a low germinating activity even though
six months had elapsed after maturity.

32.

Variation in photoperiodic response among wild rice populationof Thailand 11(By Hiko-Ichi OKA and Wen-Tsai CHANG)

In the Annual Report for 1958 (No.9: 47-48), the writers have reported
variations in photoperiodic response among Indian populations of Oryza
perennis and O. sativa f. spontanea. In 1959, similar investigations weremade using materials collected from Thailand. The results are given in
Table 1.
The data show that, in the same manner as in the Indian
materials, strains seemingly belonging to O. perennis are highly sensitive
to photoperiod, and those belonging to O. sativa f. spontanea have a relatively low sensitivity. As discussed in another paper (MORISHIMA and OKA),
however, these two species show a continuous array of intergrades. Some
1)

This work was supported by Grant RF57080 from the Rockefeller Foundation.

RESEARCHES CARRIED OUT IN 1959
Table 1.

Species

Variation in photoperiodic response in wild rice
populations collected from Thailand.

Population

Index of sensitivity (TDM)
MeanO.sativa f.
spontanea" Intermediate"

O.perennis

57

Earliest

Critical day-length

LatestMean

Earliest

71°

13"28'
13"21'

13"1·49'

13"01'

13"57'

13"30'

12"25'

W10l*
W168

86°
45°

W170

37"

13h 13'W173

45°

13h 27'

W163
W164

90°
90 0

12 h 42'12 h 44'

W171

86°

90°

12"38'

12"53'

W179

61°

n°

12"57'

13"20'

W169

90°

12 h 32'

WIn

90°

12"30'

108

3°

0°

11°

414

46°

49°

41°

504

11°

3°

19°

521

41°

40°

36°

647

15°

23°

6°

LatestO.sativa**(Cultivated)

14"04'

14"07'

14"02'

14"23'

14h 26'14 h 21'* From India.
** The difference between the earliest and the latest plants in cultivatedstrains shows the magnitude of sampling error.

populations of intermediate type showed a high sensitivity like that ofO. perennis. Regarding variation whthin populations, the data of Table 1
suggest that it is wider in O. sativa f. spontanea than in O. jJerrnnis.

33.

The impact of cultivation on populations of wild rice,
Oryza sativa f. spontanea 1)(By Hiko-Ichi OKA and Wen-Tsai CHAN})

In the Raipur District, Madhya Pradesh, India, Oryza sativa f. sfJontrznea
is a common grass, which is also found in paddy farms as a weed. The
senior writer visited this area in November, 1957, and collected seeds ofthis species from populations growing in a grass land apart from paddy
11This work was supported by Grant RF57080 from the Rockefeller Foundation.

RESEARCHES CARRIED OUT IN 1959

58

farms (I), in a patch between paddy farms (II), and in a paddy field (III),and seeds of a cultivated variety (0. sativa) from the paddy field in which
Population III was found (IV). The plants from those seed samples weregrown and investigated in Taichung, Taiwan, in a plant-to-row experiment. First, it was found that the four populations showed a continuous
range of variation in heading date, panicle length, grain weight and othercharacters. A discriminant function was computed to maximize the difference between Populations I and IV by combining those characters. Itsscore also showed a continuous variation.
It was then assumed that
Populations II and III might be due to introgressive hybridization between
wild and cultivated plants.
Secondly, the percentages of out-crossing, estimated by a biometricalmethod from vClri;mres of grain size and heading date, were found to be
25% - 30% in Population I, less than 10% in Population IV, and 10?620% in Populations II and III.
As to the degree of seed dormancy, it
was also found that Populations II and III could be regarded to be intermediate between I and IV. In parallel to this, the disturbance of habitatby man seemed to increase from I to IV. It was then hypothetically assumed that a population produced by natural hybridization between wild
and cultivated plants might acquire characters of the cultivated type inaccordance with the "pressure of cultivation" under which the population
grows.
(Published in Phyton 13: 105-117, 1959)

34.

Distribution of the glutinous gene in wild and cultivatedpopulations of rice in Thailand 1)(By Hiko-Ichi OKA)

In the Northern and North-Eastern regions of Thailand, glutinous rice
is grown in almost all paddy farms as the principal food crop. The writer
travelled through those" glutinous area" from November to December,
1958, and collected seeds from a number of wild and cultivated populations. Seven samples of cultivated glutinous rice, each consisting of 40
to 80 plants, were found to be a mixture of non-glutinous and heterozygous plants, and the frequencies of the glutinous gene varied from 97%
to 62%.
The percentage of out-crossing was estimated from the frequency of non-glutinous seeds in glutinous homozygous plants and fromthe frequency of the glutinous gene in the population. It varied from 036
to 3.6%_
1)This work was supported by Grant RF57080 from the Rockefeller Foundation.

5~

RESEARCHES CARRIED OUT IN 1959

Secondly, 14 wild populations of Oryza perennis or O. sativa f. spontanea,
surrounded by glutinous cultivated rice, were investigated in the samemanner. Six of them were purely non-glutinous, other six contained theglutinous gene with a low frequency, but the remaining two showed highfrequencies of the glutinous gene, i.e., 28% and 26%; for the former,
the percentage of out-crossing was estimated to be 44%.
This indicatesthat under certain conditions, wild rice populations can absorb genes fromadjacent cultivated plants.
Plants from the seeds of those cultivated and wild populations were investigated in Taichung, Taiwan, in a plant-to-row experiment.
Thegenetic components of variances of seed length, estimated for the wild
population with 44% out-crossing, are shown in Table 1.
Table 1.

Genetic variances of seed length in classes withdifferent combinations of the glutinous gene.

Class

d p2,

1102

++

.0567
.0499
.0488

.0680
.0906
.0059

+gl
glgl

No. of lines31
41
8

Mean(mm)
8.43
8.30
8.47

The data in Table 1 show that in inter-plant variance (!J p 2 , among lines
in the experiment), the three classes++, +gl, and glgl did not differ somuch from one another, while the glgl class had an apparently smaller
offspring variance (!J o2 , within line) than the other two classes. This may
indicate the effect of self-pollination.

35.

Population-genetic studies of wild rice of Taiwan 11(By Hiko-Ichi aKA and Wen-Tsai CHANG)

The senior writer once assumed that the wild rice found in Tao-Yuan
Prefecture, Taiwan, might be O. sativa f. spontanea (Annual Report No.6:
46-47), but its rhizomes indicate that it is rather O.jJerennis. Three small
populations, called A, Band C by the writers, can be found at present.
Seeds were taken from a number of plants of the three populations, andthe offspring were investigated in the experimental field in Taichung.
First, the results proved that the majority of plants bore the charactersof cultivated rice, such as non-shedding, heavy seed weight, etc., though
the plants in the natural habitat appear to be completely of wild type.
Secondly, with regard to seed length and seed width, the genetic vari1)

This work was supported by Grant RF57080 from the Rockefeller Foundation.

60

RESEARCHES CARRIED OUT IN 1959

ances were estimated among plants of the natural population (a p 2 ), amongline means of the offspring (a~,) and within lines (11 0 2 ).

Also the covari-ance between parental plants and offspring line means (a pp ') was estimated. The frequency of heterozygotes for genes controlling those characters(2y), relative to the percentage of out-crossing (,I.), was computed by using
the formulas given below, The results showed that within the range,1.=0.20-0.70, the frequencies of heterozygotes (2y) were much higher thanthose expected under the assumption that the population was not affectedby selection, 2y=J./l+J. (assuming that gene frequency x=~). In otherwords, the populations might consist mainly of heterozygous plants owing
to their selective advantage. In view of this fact, it was suggested thatthe populations of wild rice can absorb and preserve a large amount ofgenetic material from cultivated plant:,; and release it if sexually propage.ted.
Formulas used for computation:(1-2y)d 2+22y(1-2y)h 2 ,a;, =2=( 1--~)'2(1-2Y)d2+(1-J.)21:'-~(1-2Y)h2 ,a =( l--~ ) 2(1-2y)d +(1-J.)1:'y(1-2y)hO'~ =2[Y++(1- ~ )(1-27/)}2+,{ ~ +-~(l--~)(l- 2 y )}2
a~,

pp '

36.

2

2

,

Variation in yielding capacity among isogenic lines of rice
(By Hiko-Ichi aKA)

A variety of crop plants represents an assembly of genotypes which
respond to outer conditions in a certain manner. When a few particulargenes of the genotype are replaced by others, the genotype may in somecases be disharmonized as the whole, resulting in a break-down of thereaction-norm. In other cases, however, certain characters may be changed
without causing disintegration of the reaction-norm determined by thegenetic back-ground. For instance, early varieties of rice generally havelower yielding capacity than late ones. Taking a variety with high yielding-:apacity, if a few genes controlling the duration of growing period only could
be replaced by proper ones introduced from an early variety, the potential
yielding capacity of the genotype may be utilized to the maximum.
From the above-mentioned view-point, the writer has been engaged, withcollaboration of Mr. K. H. TSAr of the Taiwan Provincial Agricultural
College, in the breeding of isogenic lines of Taichung no. 65, a representative "Horai" variety grown in Taiwan.
The results for glutinousRESEARCHES CARRIED OUT IN 1959

61

isogenic lines were reported last year (Annual Report No.9: 34-35); in
this paper, data for early lines are presented.
A strain of Taichung no. 65 was crossed with two early varieties, andthen back-crosses were repeated seven times using the former as therecurrent parent. As the donor parent for earliness, "Tatong-tsailai"
from Northern China, and "Bozu no. 5" from Hokkaido, Japan, wereused. The B7 F s lines thus obtained were tested together with the recurrent
parent. Fifty two lines from" Tatong-tsailai " (Class A), and 5 lines from
"Bozu no. 5" (Class B) were 15 to 19 days earlier than the control(Taichung no. 65), and the other 25 lines from "Bozu no. 5" (Class C)
were 3 to 7 days earlier, though they showed the same growing habit as
Taichung no. 65. Variation in rough rice yield per plant of those lines,
tested in the second crop season of 1959, is given in Table 1. The data inthe table show that a part of the early isogenic lines may have the sameyielding capacity as Taichung no. 65.
Table 1.

Variation in yield among early isogenic lines of Taichung no. 65.
-------

Yield per plant, gm
Class- - - - - - _ .. _-

16

18
_

Control
B 7F s A
fI
B
fI
C

1
11

I

... _ - - -

2
14
3
1

I

20

22

24

11
9

3
2

7

6

28

30

I

Headingdate--,-----------_ _ - - - - . --------

-

3
16
2
5

26

No. of
lines

..

3

I 2

20
52
5
25

Oct. 8-12
Sep.21-25
Sep.21-25
Oct. 3- 7

37. Genetic variability in established varieties of rice ' )(By Kan-Ichi SAKAI)

The genetic variability in this experiment is measured in terms of heritability, i.e. amount of genetic variance as a percent of total variance.
Three rice varieties of hybrid origin in Ceylon were investigated. The
heritability values estimated for seed size characters, yield capacity, tiller
number and plant height are listed in Table 1.
It is found in Table 1 that of these three varieties, H 106 is the mostwhile H 105 is the least heterogeneous. A number of lines were selectedfrom different yield levels in each variety, and they were planted in thefollowing season. The correlation between yields of the two season iscomputed (Table 2).
1)This study was conducted in the Division of Botany, Department of Agriculture,
Ceylon.

62

RESEARCHES CARRIED OUT IN 1959
Table 1.

Heritability values of different quantitative charactersin three rice varieties.

Seed length
Seed width
Yield
Tiller number
Plant height
Table 2.

H 105

H 106

H 501

21.85
11.51
11.58
0.84
30.64

82.51
24.95

37.95
28.55
20.19
0.00
47.06

38.17
19.72
66.61

The total, between-classes, and within-classes correlation
coefficients between yields of two consecutive seasons.
CorreiaLion coefficients

Total
Between-classes
Within-classes

H 105

H 106

H 501

0.78110.9951
0.34800.60050.9319-0.01400.55850.68900.2759

Table 2 shows that in general, the yields of the same lines in two
consecutive seasons are positively correlated. The correlation is very highif different classes are compared, but very low if comparison is made withinclasses. On the basis of these findings, it is concluded that:1) Established varieties of rice, a plant which is believed to be an
autogamous plant, are not always genetically pure. They are more likely
mixtures of different lines, at least with respect to polygenic characters.
2) In view of this, an established variety of rice may be capable ofaltering its performance during its maintenance and propagation.
3) A suggestion is made that seed of high genetic purity could be
produced by improving the scheme of seed farm operation by introducing
simplified performance tests accompanied by elimination of inferior lines.
Heritability tests should also be included in the scheme.
Details of the present study will be published at another opportunity.

38.

Variability of seed size characters in wild ricel)(By Kan-Ichi SAKAI, Takashi NARISE and Shin-ya IYAMA)

Variability of seed size characters was investigated for several wild ricel)
The main part of this study was conducted at Peradeniya, Ceylon, while theremaining part was done at Misima, supported by Grant RF57080 from the Rockefeller Foundation.

63

RESEARCHES CARRIED OUT IN 1959

populations of Oryza perennis collected in five localities on the west coastof Ceylon. Measurements were taken for length and breadth of seed,mean values of which are presented in Table 1.
Table 1.

Mean values of length and breadth of seed in six
populations of wild rice.
c-==,,-~'-c===c==-,,= --------~-.

Population
Ky
Nt
Th-(I)
Th-(2)
Kd
Md

,..
D

Locality---~.-~==-~_~_

N~e~~u~~~~I-~~:d length--

Kirimetiana
Nattandiya
Thumodara
Thumodara
Kudawala
Madampe

50
50
50
27
50
50

Seed breadth

7.96±0.51
7.47±0.49
7.15±0.83
7.62±0.33
7.93±0.27
8.04±0.37

2.45±0.23
2.67±0.29
2.85±0.14
2.31±0.13
2.14±0.1O
2.34±0.16

Genetic and environmental variance components as well as genetic andenvironmental correlations were estimated for each population, assumingthat within-plant variation or covariation is environmental while betweenplant variation or covariation is the sum of environmental and genetic
variations. The proportion of genetic variance is presented in Table 2.
Genetic and environmental correlations between seed length and seedbreadth are also presented in the same table.
Table 2.

Genetic variability and genetic and environmental correlations
for seed size characters of wild populations of rice.
Genetic variance

Population

I

Mean

----

Seed length Ii Seed breadth

Correlations

Enviro~mentall

Genetic------Ky
Nt
Th-(I)
Th-(2)
Kd
Md

0.1836
0.78920.96880.78920.84830.68800.58350.64900.74230.34560.7026
0.5598- - - - _ ..

_-0.38360.7191I

0.8556
0.56740.77550.6239-------

0.00510.0197-0.05440.29610.17500.2416

0.09350.0267-0.0005
0.6666
0.44100.3039-~-----

It is found from Table 1 that seed length as well as seed breadth differ
markedly from population to population, the difference being often statistically significant. Table 2 shows that most populations are very heterogeneous
so far as size characters are concerned.
The same investigation will be continued with other populations.

64

RESEARCHES CARRIED OUT IN 1959

39.

Variation in anthe:.is in wild rice populations (Oryza sativa
f. spontanea) collected in India and Ceylon!1(By Takashi NARISE and Shin-ya IYAMA)

Number of days from seeding to flowering of four wild rice populations(Oryza sativa f. spontanea) collected in India and Ceylon was investigated.
The places where the material was collected are as follows.
TR
Trichur
Kerala State, India
CN
CanningWest Bengal State, India
CT-b
Cuttack
Orissa State, India
PL
PuttalamWest coast of Ceylon
Progeny lines of plants collected at random in the wild populations weregrown in an experimental field at Peradeniya, Ceylon. Seeds were sownin the nursery on April 8, 1959 and the seedlings were transplanted intoa paddy field on May 6. Each line contained two to ten plants on which
flowering date observation was made. Within-population variability of lines
with respect to number of days from seeding to flowering is shown in
Table 1.

Frequency distribution of number of days from seeding toflowering in four populations of O. sativa f. spontanea.
No. oflines

Population

1~0 1~0 1~0 140 1~0 160 1~0 180 1~01
120

130 140

Average

150 160 170 180 190 200
1

CN
CT-b
TR
PT

19
19
19
31
Table 2.

8

12
9

7
3

6

9

15

5

159.4 days
163.4
122.5

10

1

1 I 164.5

Analysis of variance of number of days from seeding
to flowering of four wild rice populations. 1959.
~~~~.- - - - _ . -Source ofvariation

!

I

I.

d.f. I Mean squareI·
Total-- -Ii 372 . --..
I

--,-1

Estimate of
the componentof variance

Expectation of meansquare_!

Population

3

30,621.818**

Lines
population
Within lines84

241.401**

"82

285

100.375

rJ w 2~ithinA

33.514A-

1

100.375" __- - - -** Significant at 1 % level.
!IThis work was supported by Grant RF57080 from the Rockefeller Foundation.

RESEARCHES CARRIED OUT IN 1959

65

Table 1. The results of analysis of variance is presented in Table 2.
traclass correlation coefficients were computed as follows.
ds 2

Between strains within populations:

+d w2

0.250

+ ds 2 + d w 2

0.710

ds

2

d p2

Between populations:
d p2

In-

As Table 2 shows, between population variation and between lines within
population variation were statistically significant, though the latter was
relatively small.
The result of this experiment indicates that wild rice populations arehighly differentiated with regard to flowering time.

40. Further note on natural crossing in wild ricel)(By Kan-Ichi SAKAI and Takashi NARIS E)

Occurrence of natural crossing in wild rice was measured by means ofbiometric analysis of seed width. Wild rices investigated were threegeographical races of Oryza sativa f. spontanea, collected in India. One
Ceylon variety of cultivated rice, O. sativa L., was also investigated forcomparison. Lines were grown from plants collected at random andanalysis of variance of seed width was made (Tablen

Table 1. Within and between lines variances and the estimated percentage
of natural crossing in wild rice populations. One variety, H 106,of cultivated rice is also included for comparison.
H 106
variety---------

O. sativa f. spontanea
TrichurCanning

Cuttack-----

Within-line variance0.00220.01140.01790.0266

Between-line variance0.0596

0.02670.04310.1413

Percentage of outcrossing

3.60

33.91

31.93

-------_._~

16.55------

Percentages of outcrossing listed in Table 1 suggest that wild rice plants
are disposed to more frequent cross-pollination under natural circumstances
than domesticated varieties.

11
This study was conducted in Peradeniya, Ceylon, supported by Grant RF57080
from the Rockefeller Foundation.

66

RESEARCHES CARRIED OUT IN 1959

Competition experiment with wild rice l )41.

(By Takashi NARISE and Kan-Ichi SAKAI)

Oryza perennis collected at Cuttack, India and O. sativa f. spontanea at
Illuppaiyadichchenai, Ceylon, were tested for their competitive ablility
against two cultivated varieties of rice in Ceylon. O. perennis was tested
in the Maha (winter) season of 1958/59, while spontanea was tested in two
consecutive seasons of Maha 58/59 and Yala (summer) 1959. The test
varieties used in this experiment were Murungakayan 302 and H 105.
Plant weight and number of tillers were compared between pure-standand mixtures with each of the test varieties. Experiments were all conducted with three replications.
nf ,-l"t" ",hnU7p,-lulith...
-An" 1v",;", nf v"ri"nrp.
,. -- th"t thp pffprt nf rnITlnptit;nn.r-'....,~--_.-.J

~--

~~

~~

~~~~~-

~

~~~-

~~~

A~

_ _ .~'-'

--

~

"'

test varieties was highly significant for plant weight as well as numberof tillers in perennis but not for the former in spontanea. Mean valuesof tiller number and plant weight in pure-stand and mixtures with thetwo test varieties are presented in Table 1.
Table 1.

Number of tillers and plant weight of two wild species of ricein pure-stand and mixtures with two test varieties.
Number of tillers

per;,ennts
Maha

Plant weightper:spontanea
Maha

Yala

I

Mixture with
M 302

Maha

Mean

.

Pure-standspontanea

enn~s

I

Maha

Yala

Mean

--------

9.38

10.62 22.37

16.50

7.05

32.42 79.77

56.10

2.20

3.71

15.71

9.71

6.42

11.16 72.52

41.84

2.95

17.90

10.43

5.45

7.54 93.07

50.31

,

Mixture with
H 105------

I

1.97! -------!

!

Table 1 shows that number of tillers as well as plant weight of wild ricedecrease if they are grown in competition with the cultivated varieties usedin this experiment. It means that wild rice is a weak competitor against
the test varieties.
In another experiment dealing with competition among a number ofcultivated varieties of rice, it was observed that Murungakayan 302 andH 105 could be taken as representatives of competitive ability of thecultivated varieties of Ceylon. It can be concluded then that wild rice11
The present study was carried out in Peradeniya, Ceylon, supported by Grant
RF57080 from the Rockefeller Foundation.

RESEARCHES CARRIED OUT IN 1959

67

may be very weak in competitive ability in comparison with cultivatedrice. This result is of interest from the viewpoint of the evolution of wild
plants in the presence of cultivated varieties.

42.

Propagating capacity and competitive ability of the F , hybrid
between upland rice and "red rice".
(By Shin-ya IYAMA)The field of upland rice is often contaminated by the coarse "red rice"
variety which has long been removed from our list of commercial varieties
but is still surviving as a weed in rice fields. Spontaneous hybrids may
arise between the red rice and a commercial variety of upland rice. The
present experiment has been undertaken for the purpose of elucidating thefate of those hybrids in the upland rice population.
1) Propagating capacity of the F , hybrid.
Phenotypic comparison of "red rice", two varieties of upland rice,
N6rin Machi Nos. 8 and 18, and their F , hybrids are shown in Table 1.
Table 1. Several characters of the F , hybrids between upland rice and"red rice" (Norin Mochi No. 8 X" red rice" and Norin Mochi
No. 18 x" red rice") in comparison with
their parental varieties.
Fertility
Plant I Panicle No. of: Total Panicle I No. ofheight I length I panicles' weight I wt. per seeds per:.
Iplant I plantpollenseed_- - _.. _ - - - - - - - - -cm
Norin M. No.8
90.7
F , (8xR)*
126.4
Norin M. No. 18 82.5
F , (18xR)**
I 127.7"Red rice"
84.4------'-______

I.... _ -

---

-

I

cm
20.0
22.2
19.7
22.9
16.3

7.4
15.4
7.4
15.7
18.9

---- -

-

g
22.3
56.2
23.0
60.7
32.6

-----

g
9.8
19.5
8.0
17.1
8.9

0/

%

/0

I

333.9
357.6
232.4
314.2
324.1

98.2
43.7
98.4
33.6
97.1

,

58.6
80.9
48.2
85.2

* F , : Norin Mochi No. 8 X " red rice".
** F , : Norin Machi No. 18 x" red rice".

As Table 1 shows, the F 1 hybrids produce more seeds than the parental
upland rice varieties although no great difference from "red rice" isrecorded. It is rather surprising that the number of seeds of the hybrid
is not less than that of its parents because of high hybrid sterility.
2) Competitive ability of the F , hybrids.
The F 1 hybrid plants were grown in a row together with their parental
upland rice variety planted in alternate hills. The characters of the F 1
and the parental plants in the competition plot were compared with those68

RESEARCHES CARRIED OUT IN 1959

in the pure-stand plot. The difference between the two plots would be anindex of competitive ability. The result of analysis of variance for competitive ability on a logarithmic scale is presented in Table 2. Gain orloss in the characters due to competition is shown in Table 3.
Table 2. Analysis of variance of competitive ability of F 1 hybrids
and parental upland rice varieties (logarithmic scale).

Source of variation

d.f.l-panicI~-TotalMea_;_a:_~c_~:r~I number 1 weight ,wt.jplant

Replication
StrainParents vs. F,'sParental group+
Interaction

Paniclelength1---' -x 10- 3- - -2.011,24::i.192,30*;;:1 .250,31**1 .388,38**1 2.0398.001,34.001,96

1
1
1.554,79**1.722, 96**r 129,82**[ 6.1156*.015,77.017,38 I .025,02 . .0291.006,35· .010,65.010,31.1548.005,13.000,43.000,31_~

Error _

iI .023,11

Plant
height.000,72

1 .1906

1

1_i __'0_1~7 _'_.0_0_9,_3_81_~026, 4_6__.568_1_'----.0_01,35
1+ (Nor in Mochi No.8 and its F 1 ) vs. (Norin Mochi No. 18 and its F 1).
* Singnificant at 5% level. ** Significant at 1% level.
Table 3.

Increment due to competition (difference between mix-plantedplots and pure-stand plots). Measurements forpure-stand plots are shown in Table 1.

Plan~c~ 1 P~:~cle -Panicle I Total" 1 Panicle_ _ _ _ _ _ _ _ _ I..:t.jpla~l---.:.eight -,-I_n_u_m_b_e_r_
Norin Mochi No. 8
F 1 (8xR)
Norin Mochi No. 18
F 1 (18xR)

1-

4.6g i- g
7.2 ·I

2.3

height,----= 6. 2cm l----=
1

1

18.2
- 5.0
__1_4_.1

47.3
-11.8

8.8
3.8

4_804----'-_ _7_._6

length
cm
0.8

I

6.7
4.8

1_~1

I

0.7
1.7

__ ~_

Inspection of Tables 2 and 3 leads us to conclude that: (1) Differencein competitive ability of any of the F 1 hybrids and any upland rice variety
is statistically significant. (2) F 1 hybrids show a marked increase as aresult of competition with the parental varieties, while the latter show asignificant decrease.
In conclusion, it may be expected that spontaneous F 1 hybrids fromnatural hybridization would be favored in an upland rice population due
to their strong competitive ability and heterosis in spite of high hybrid
sterility.

RESEARCHES CARRIED OUT IN 1959

43.

69

Seasonal sterility of rice in Ceylon ll(By Kan-Ichi SAKAI)

Three varieties of rice grown in Ceylon were planted in pots in a plantcage on the 17th of every month from December, 1957 through November,
1958. Each plot consisted of 9 plants of each variety. The seed sterility
averaged on a 9 plant basis for every plot is presented in Table 1.
Table 1.

Seed sterility of three varieties of rice planted at one monthintervals from December 1957 through November 1958.
Seed sterility (%)- - - --------MurungakaMurugan
Mean
yan 302
Samba 3081

I

Treatment
numberDate of - - - - - planting
Mas M-24'II---- ' - -

- -1

17/12/57 I

37.31

15.97

28.10

2

17/ 1/58 I

22.98

9.59

24.77

19.11

3

17/2/58 i
23.96
17/3/58 '24.28

8.28
9.17

21.48
22.63

17.91
18.69

40.18

27.02

44.06

38.75

42.10

20.31

18.77

27.06

36.01

13.01

26.21

25.08

11.98

2.50

22.00

12.16

29.22

5.40

53.48

4

5
6
7

8
9
10
11
12
Mean

17/4/58 i! 17/5/58
17/6/58
17/7/58
17/ 8/58
17/ 9/58~~;~~;:: :_ _ 30~76

1_

29.88

33.98__
I

14.52

L

27.13

29.37
26.37

14.37
27.59

------~---------

The following facts are pointed out by this experiment:
1) Seed sterility is especially high in plants planted between April andJune and between September and October.
2) Varieties differ with respect to the occrrence of seed sterility.
The cause of this seed sterility is uncertain. However, the facts describedabove are of interest for two reasons. One is that rice plants are usuallyplanted in April and September in Ceylon. The other is that the occurrenceof seasonal seed sterility seems to be genotypically controlled.

1)
This experiment was conducted in the Division of Botany, Department ofAgriculture, Ceylon.

70

RESEARCHES CARRIED OUT IN 1959

44.

Studies on a laboratory method of testing salinity resistancein rice varieties!)(By Kan-Ichi SAKAI and P. M. RODRIGO)" Salt resistance" of rice plants includes two aspects, the physiologicalresistance of the plant against saline and the overall resistance of theplant as shown by its performance under field conditions. The present
study has been undertaken to detect a laboratory method for testing thephysiological resistance of rice varieties against salinity. A number ofvarieties of rice were grown in pots without any fertilizer or manure. At
the end of 40 days the plants were removed from the pots and transferred
to conical flasks of water for 48 hours. Afterwards, the water was replacedwith saline solutions, the salt useu being unrefined common salt. The
plants were left with their roots immersed in saline solutions for a definite
number of days whereupon the evaluation of resistance was made bygrouping the plants according to the damage caused. The damage was
first observed in the rolling up of the leaf tips, followed by rolling up of
the entire leaf blade, yellowing and wilting of the entire plant. The
degree of damage was classified from 0 (least damaged) to 5 (most damaged).
The experiment started by treating plants with 20%. 10%, 5% and 0%
saline solutions. It was found in this experiment that even 5% concentration was too high to detect varietal differences. In the second experiment,
4, 2, 1 and 0% saline solutions were used. It was found that 2% wasstill too high to allow an effective comparison among different varieties.
In the third experiment treatment was made with 1.5%, 1%, 0.5% and
0% saline solutions and the evaluation of damage caused was made twice,
2 days and 7 days after treatment. Table 1 presents the correlation coeffiTable 1.

Correlation coefficients between the 1st and the 2nd
evaluation in the 3rd experiment.
iPercentage of salt in solution

Degrees of freedom
1 - 1 . 5 % - - 1.0%26______ L

0.5581**0.7131**

0.5%
0.6390**_** Significant at the 1% level of probability.

dents between the records of the first evaluation made on the third day
and those of the second evaluation made on the eighth day of the third
11
The present study was conducted in the Division of Botany, Department ofAgriculture, Ceylon.

RESEARCHES CARRIED OUT IN 195971

experiment.
The used varieties were divided into two groups: salt-resistant and nonresistant. The latter group includes varieties whose resistance to salineconditions is either in doubt or unknown. A chi-square test was conductedon the relation between the results of the experiment and the two groupings,
resistant vs. non-resistant (Table 2).
Table 2.

Chi-square test.
Salt solution

X2

1st evaluation

Probability- - - _ ...

1.5%

0.5,;;

1.60720.20-0.50

1.8046
0.10-0.20_--_.--

2nd evaluation!
!
I

X2

Probability

2.02860.02-0.05

11.3000
0.01--------

2.02550.10-0.20

Tables 1 and 2 show that the most suitable of the three concentrationsof salt solution for discriminating between resistant and non-resistantvarieties would be the 1% concentration and also that evaluation on the
8th day after the commencement of the treatment would give better results
than evaluation on the 3rd day.
Data obtained in the second and third experiments include six treatments,
4%, 2%, 1.596, 1% (twice) and 0.5%. Analysis of variance, estimation of
repeatability, and chi-square test of the collective data were conducted.
The results of these analyses can be summarized as follows:(1) Varietal differences in response were quite distinct.
(2) The salt resistant variety group differed significantly from the nonresistant group.
(3) Differences between varieties within the" non-resistant" group werehighly significant.
(4) Differences between varieties within the resistant variety group were
not always significant.
(5) The repeatability of evaluation was 54% if treatment was madewith a 1-1.5% solution of salt.
(6) The degree of damage observed in 1-1.5% salt solution is likely toshow better agreement with the so-called salt-resistance than the damage
caused by 4, 2 or 0.5% salt solutions.

RESEARCHES CARRIED OUT IN 1959

72

45.

Variation in susceptibility to Piricularia disease inwild rice populations!)(By Takashi NARISE)

Three populations of wild rice, Oryza sativa f. spontanea, were investigated for inter- and intra-population variability with respect to resistanceto the blast disease, Piricularia oryzae. Localities where wild rice populations were collected are: Chinsurah (West Bengal, India), Samalkot(Andhura, India), and Puttalam (West coast, Ceylon). Plants collected inthese localities were harvested on an individual plant basis, and progenies
were grown in Peradeniya, Ceylon in the summer of 1959. Susceptibility
to the disease was tested by the leaf-sheath method using the leaf sheathof next to last leaf. Testing was done twice for each individual plant.
The strain of Piricuiaria oryzea was collected in Ceylon in 1958. Averagesusceptibility value and genetic variability of each population are presented
in Table 1.
Table 1.

Average value and genetic variability of susceptibility to

Piricularia disease in populations of wild rice,
O. sativa f. spontanea
Population

LocalityB
1'-4Chinsurah
SamalkotPuttalam

Averagesusceptibility*

IIGenetic variability(%)-_--3.212985.64
0.9991
99.56
3.4342
95.81.. _ - - . .

PT.

*) The estimate for the most suseptible plant being 5.0.
**) Genetic variance expressed in percent of total variance.

Table 1 shows that populations differ markedly with regard to diseasesusceptibility. The T -4 population collected in Samalkot is as a whole
highly resistant to the disease, while those collected in Chinsurah andPuttalam are highly susceptible. It is also found that within population
variability is very high in all populations.
Partitioning of component of variance into environmental variance (11}),
between progeny plants within lines variance (11[2), between lines within
populations variance (l1s2), and between populations variance (l1p 2) was made,and variability on each level was estimated as follows:
Between progeny plants variability:

11[2l1 e 2+ 11[20.9224

11
This experiment was conducted in Peradeniya, Ceylon under the kind guidanceof Dr. Yoshio Takahashi, Professor of Yamagata University, Japan. It was supported by Grant AF57080 from the Rockefeller Foundation.

RESEARCHES CARRIED OUT IN 1959

Between lines variability:
0'.2

Between population variability:+ 0'£2 + 0'8 273

0.13600' p2- - - ._
- - 0.7333
20',2 +0'£8 +0'8

+ 0' p2

These figures suggest that plants in wild population are highly heterozygous
with respect to disease susceptibility, and the inter-population variability
is very high.

46.

Susceptibility of nullisomic wheat dwarfs and their respective
gigas-plants to leaf rust, Puccinia triticina(By Seiji MATSUMURA and Keizo KATSUYA)

Dwarf plants possessing 40 chromosomes (20n), found among the offspring of the pentaploid hybrid, Triticum polonicum x T. Spelta, are nullisomics, deficient in a chromosome pair of the D-genome. Depending on
which of the a~g D-chromosomes is missing, they are called a~g-dwarfs.
The respective gigas-plants with 42 chromosomes found among the offspring of nullisomics are called a--g-gigas.
They also may be termed
D-nulli- and AB-tetrasomics.
In order to examine the susceptibility of nullisomics and their gigasplants to leaf rust, a~g-dwarfs and a~g-gigas were tested at the firstleaf stage with Puccinia triticina 21 B.
One parent, T. polonicum, wassusceptible, while the other, T. Spelta, showed resistance to rust. Because
all e-dwarfs and their gigas-strains were susceptible, it has been concluded
that the gene for resistance is located on the e-chromosome of the Dgenome. A strain of g-dwarfs was susceptible, but a gigas-strain originating from another g-dwarf was resistant.
The c-dwarfs and their gigasplants exhibited resistance or moderate resistance and b-dwarf and its
gigas-plants were resistant. Though a-dwarf was resistant, among a-gigasIt has been asplants, resistant and susceptible strains were observed.
sumed that the supernumerary chromosomes of resistant and susceptible
a-gigas-strains were different. Among d- and f-dwarfs resistant and susceptible strains were found and also the correspondent gigas-strains showed
resistance and susceptibility, respectively. We supposed that more genes
for susceptibility are located on the chromosomes of the A- and B-genomes,
in addition to the epistatic genes of the D-genome.

74

RESEARCHES CARRIED OUT IN 1959

47.

Recovery of chlorophyll content in SOine mutants in einkorn
wheat, Triticum monococcum fiavescens(By Tar6 FUJII)

Viability of the virido-albina mutant is poor, but the chlorophyll content
recovers almost normal when the plants are placed in the phytotron (d.
Ann. Rep. No.6). On the other hand, double recessive plants viridoalbina-basi-viridis II look like albina and, even in the phytotron diedwithout developing chlorophyll, while both parents have the ability to recover (d. Ann. Rep. No.8).
Moreover, the new chlorophyll mutants,
basi-viridis III, virido-albina II, and xantha-alba I, obtained from irradiation experiments, which have little chlorophyll at the seedling stage, also
have shO'.vn the ability to develop almost normal chlorophyll content.
From these findings, it appears that in certain chlorophyll mutants therecovery of chlorophyll content might be initiated in the presence of asmall amount of chlorophyll in the leaf tips.
In an experiment, the green tips of the leaves were cut off and theplants were placed in the greenhouse and in the phytotron at seedlingstage or later, when the young plants had developed 5 - 6 leaves.
The
batch in which the green parts were removed did not grow, and the plants
died after about 2 weeks, while the batch containing uncut plants continued
to develop.
On the other hand, recovery of the chlorophyll content in the viridoalbina mutant in agar culture with WHITE'S solution to which 3 percent
sucrose was added was faster than in soil culture. After 20 clays in agar
culture the plants recovered to normal green, while it took about one
month for the recovery in soil in the phytotron. Thus, it seems that agar
culture is more suitable for increasing the chlorophyll content than soil
culture. But, the cut batches of virido-albina died also in agar culture.
Albina mutants and albina like plants obtained from the cross between
virido-albina and basi-viridis II were also sown on agar, but they neverdeveloped any chlorophyll. It is well known that chlorophyll and radiantenergy are the essentials of photosynthesis, the most important process
for plant growth. The virido-albina mutant, mentioned above, could notsurvive when the green parts of the leaves were cut off.
A smallamount of chlorophyll was necessary for priming the process of further
chlorophyll development (d. FUJII 1959, Jap. Jour. Genet. 34).

RESEARCHES CARRIED OUT IN 1959

48.

75

Amount of amino acids in the chlorophyll mutants of einkorn wheat(By Yukio ONO and Taro FUJII)

Amino acid content in several chlorophyll mutants of Triticum monococcum fiavescens was examined at the seedling stage. Four albinas, one
chlorina, and one virido-albina, which has the ability to recover the chlorophyll content, were used in this examination. Normals were used as control. Free amino acid was extracted from young leaves with 75% ethanol,and the several amino acids were separated by two dimensional paper
chromatography. Seven kinds of free amino acids, namely aspartic acid,gultamic acid, glycine, alanine, glutamine, phenylalanine and leucine,were detected. Four albinas and one virido-albina had all of them, whileleucine and alanine were not observed either in normals or chlorina. Albina
and virido-albina mutants showed larger amounts of all amino acids than
the normals and chlorina.
Cysteine, histidine, proline, and methionine
were not observed in normals and chlorina, but in albina strains only
traces of these were found.
Moreover, non-identified spots (a and b in
Figure 1 and 2) were observed in all strains, and the a-spot size waslarger in normals than in albina.

rI:\

~

Glua

~

Pile-at

AtJ9-2 Q~-~

Fig. 1.

G.11I

PhenolFree amino acids in the normal strain.

Kinds and amount of organized amino acids were also determined by
the same chromatographic method after hydrolysis of fresh leaves. About
14 kinds of organized amino acids were observed in normals, while only

RESEARCHES CARRIED OUT IN 195976

10 kinds were found in albina. They occurred in larger amounts in normals than in albina. Leucine, glutamine, alanine, and the non-identified
spot (a) contained in free amino acids, were not observed in organizedleu

8

Pke-1l1r>..

~

Ala. (h)

u~D
'~
&/u

Fig. 2.

,~ PlleHol
Free amino acids in the albina strain.

amino acids. Therefore, they may be faster transferable compared withother amino acids. From these results, it appears that the albina strains
have a larger amount of free amino acids and a smaller amount of organizedamino acids than the normals.

49.

Susceptibility of wild rice to blast fungus, Piricularia oryzae(By Keizo KATSUYA)

In order to examine the susceptibility of wild rice to blast, we tested
a strain each of Oryza minuta and O. officinalis, 3 strains of O. perennis
and O. sativa var. spontanea and 27 strains of O. glaberrima in the greenhouse with 2 strains of Piricularia oryzae, namely P-2 and 54-04, obtained
from the National Institute of Agricultural Sciences.
For each trial 10
plants were kept in pots to which ammonium sulphate, calcium superphosphate and potassium sulphate were added. They were inoculated by spraying on the third or fourth leaf or by injection into one of the fourth
to sixth leaves. The degree of susceptibility was determined by the typesof lesions formed on the leaves. Blast readings were made on infected
leaves 5 to 10 days after inoculation. The tests were repeated 2 to 3 times.

77

RESEARCHES CARRIED OUT IN 1959

o. minuta (W0016) was resistant to the two strains of blast fungus.
O. officinalis (W0046) showed resistance when sprayed and resistance ormoderate susceptibility when injection was used. All strains of O. glaberrima were resistant to the two blast strains when sprayed and resistantor moderately susceptible when injection was used. A strain of O. perennis (W0034) was very susceptible to the two strains of blast fungus whensprayed or injected. A strain of O. sativa var. spontanea (W0150) was
moderately susceptible or very susceptible to the two blast strains when
inoculated by injection or spraying. Some strains of O. sativa var. spontanea (W0106, W0123) and O. perennis (W0032, W0033) exhibited resistancewhen sprayed and resistance or moderate susceptibility when inoculated
by injection. Some strains of O. perennis, O. sativa var. spontanea andO. glaberrima exhibited considerably different susceptibility within thesame strain.

50.

Susceptibility of the albina mutant of einkorn wheat to leaf andstem rusts, Puccinia triticina and P. graminis(By Keizo KATSUYA)

The susceptibility of the albina mutant of Triticum monococcum flavescens to stem and leaf rusts was tested in the phytotron (20 e daytime,
15°e at night) at the second leaf stage with Puccinia triticina 21 Band P.
graminis f. sp. tritici 21. At first albina seedlings were kept in a sowing
box and then they were placed in water culture with WHITE'S nutrient
medium (1943) to which 3 percent sucrose was added. They were inoculated by brushing. Rust readings were made 7 to 16 days after inoculation.
The degree of susceptibility was determined by the types of lesions formedon the leaves. The tests were repeated 2 to 3 times.
Albina exhibited susceptibility to leaf and stem rusts, while the normals
were resistant to the former and susceptible to the latter. The uredosoriof stem rust on albina were much larger than those on normal plants.
Thus, susceptibility of host plants to leaf and stem rusts belonging to
obligate parasites is not necessarily connected with quantitative differencesin chlorophyll.
A difference in another component may be the cause ofa higher susceptibility of albina to leaf rust. Studies concerning aminoacids await further experiments.
051.

Cytogenetic studies of the genus Nicotiana XII.
(By YO T AKENAKA)

The reduction division in PMC's were studied in 5 interspecific hybrids:

78

RESEARCHES CARRIED OUT IN 1959

N. debneyi X N. otophora, N. knightiana X N. rustica, N. repanda X N.
palmeri, N. bigelovii X N. megalosiphon, and N. trigonophylla X N. palmeri.
1) F 1 of N. debneyi (n=24) X N. otophora (n=12)
So far as I know, no report on this hybrid has been published. At MI
in PMC's of this hybrid, the number of bivalents ranged from to 6, withthe mode at 2. The frequency of PMC's with one bivalent and 3 bivalents followed that of those with 2 bivalents. PMC's with or 4 bivalents
were occasionally found but those with 5 and 6 bivalents were very rare.
Considering the small number of bivalents, it is difficult to determine
whether the chromosomal affinities are allosyndetic between the genomesof the parents or autosyndetic between the subgenomes of N. debneyi
which may be of amphidiploid origin.
2) F 1 of N. knightiana (n=12) X N. rustica (n=24)
So far as I know, this hybrid also has not been reported previously.
MI in PMC's of this hybrid showed in most cells both 12 univalents andbivalents, except for the very rare occurrence of one trivalent. At AI in
PMC's, one or two chromosome bridges were occasionally found.
N.
knightiana is considered to be closely related to N. paniculata, which isassumed to be one of the parents of the amphidiploid N. rustica. Accordingly the 12 bivalents shown in this hybrid may be supposed to becaused by the affinity between the genomes of N. knightiana and of N.
paniculata which represent one of the two subgenomes in N. rustica. But
it is thought that the genomes of N. knightiana and of N. paniculata in
N. rustica are somewhat differentiated by inversions and other irregularities. The chromosome bridges at AI may be caused by such differences.
3) F 1 of N. repanda (n=24) x N. palmeri (n=12)
At MI of PMC's of this hybrid, the number of bivalents ranged fromto 9, with the mode at 6. The frequency of PMC's with 3 and 5 bivalents followed that of PMC's with 6 bivalents. PMC's with 2 and 5 bivalents were frequently found but those with 0, 1, 7, 8 and 9 bivalents
were rare.
In the same hybrid, Goodspeed (1954) found that the number of bivalents ranged from 4 to 10, with the mode at 6, and assumed that N. palmeri
was a descendant of one of N. repanda's progenitors, though there is notcomplete pairing between the genome of N. palmeri and one subgenomeof N. repanda, either or both causing the accumulation of products oftranslocations and other irregularities within genomes of the two species.
Although there are some differences between Goodspeed's observations
and mine, his speculation may be generally correct.
4) F 1 of N. bigelavii (n=24) x N. megalosiphon (n=20)
At MI of PMC's of this hybrid, bivalent chromosomes were very rare.

°

°

°

RESEARCHES CARRIED OUT IN 1959

79

The number of PMC's with 0, 1 and 2 bivalents per cell were 71, 25 and
7 respectively. And in many PMC's a restitution nucleus was formed
without the completion of first division.
It is difficult to speculate on the relationship between N. bigelovii which
is an American species and N. megalosiphon which is an Australasianspecies.
Kostoff (1941-43) has studied this hybrid; my results agree well with his.
5) F, ,of N. trigonophylla (n=12) x N. palmeri (n=12)
At MI of PMC's of this hybrid, 12 bivalents formed by pairing of chromosomes of similar shape and size were observed in all cells. At AI, allbivalents divided at the same time.
Not even a chromosome bridge ora stray chromosome was observed. Also all PMC's regularly completed
the second division and produced normal pollen grains.
Indeed, thishybrid was fertile and produced many seeds. Accordingly, N. trigonophylla
and N. palmeri may be grouped together, though they differ in flower size.
N. palmeri was considered by East (1928, 1932) to be a variety of N. trigonophylla, but MCCRAY (1932) belived it to be a different species. The
cytological data presented by KOSTOFF (1941-43) agree very well with myresults.

52.

Structural changes of chromosomes of Allium scorodoprasum
var. viviparum(By YO T AKENAKA)

The somatic chromosome number of A. scorodoprasum var. viviparum
has been determined as sixteen by many investigators, e. g. KATAYAMA(1928), MORINAGA and FUKUSHIMA (1931), TAKENAKA (1931), Y. ONO (1935) andKURITA (1951). In a previous paper (1931), the present author hinted at a
possibly hybrid origin of this species on the basis of his observations ofchromosome rings at meiosis and two somatic chromosomes of markedly
different shape and size. He further studied in detail the meiosis of thisplant, as reported in the annual report No.4 (1954). Since then three newvarieties have been brought from Formosa.
In the present paper, theirchromosomal behavior is compared with that of the variety widely grownin Japan.
(1) From the measurement of the length of root-tip chromosomes, I candistinguish six groups, a, a', b, b', c and e, each consisting of two similar
chromosomes. A Formosan variety, Nankotsu, showed no significant difference from the Japanese variety used for comparison.
The f chromosome was single; the above two varieties showed the samearm length ratio.

80

RESEARCHES CARRIED OUT IN 1959

The other chromosomes, d, d', and g, were also single and were comIn their relative length, the Japanese andposed each of three parts.
Formosan varieties showed a difference.
(2) The pairing of chromosomes at MI of PMC's was studied in theJapanese and two Formosan varieties, Nankotsu and Hokunin. In the Japanese variety, formation of five bivalents and one ring-shaped hexavalent
was found in most cells, such configurations reaching 73% of the total
number observed. In Hokunin variety, the same configuration occurredin 47% of observed cells, but cells with four bivalents and one ringshaped octovalent were also found with 22% frequency. In Nankotsu
variety, cells with four bivalents and one riI!-g-shaped octovalent were frequently found, reaching 49% of the total cells observed, but cells with 3bivalents and one ring-shaped decavalent were round as rreL[ueIlLly as 24%.
The writer has assumed a hybrid origin of Allium scorodoprasum var.
viviparum (1931, 1954). Under this assumption, it may be that this species
has changed or is changing its chromosomal structure since its establishment. The changes might have taken place in d, d', and g chromosomes,
because the relative length of three parts of these chromosomes of theabove two varieties is different.

53.

Selection for high content of nornicotine(By Kazuo FURUSATO and Akira MIYAZAWA)

Synthetic tobacco obtained from the cross Nicotiana sylvestris x N.
tomentosiformis was crossed with the commercial variety Bright Yellow.
The hybrid was selfpollinated for five years. Yearly selection for nornicotine finally yielded 18 plants with high nornicotine and very low nicotine
content. These will be used in our tobacco breeding program.

54.

Influence of environment on the fertility of tobacco(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.

RESEARCHES CARRIED OUT IN 1959

55.

81

A case of different results from reciprocal crosses(a preliminary report)(By F. A. LILIENFELD)This preliminary report is confined to a few essential facts. They arebriefly outlined as follows.
1. Two strains, named D and Sl), of two ecotypes of Medicago truncatula Gaertn., both with normal chlorophyll content, were crossed reciprocally.
2. The F l seedlings obtained from the reciprocal crosses were strikingly
different in the coloring of the cotyledons. When D was the mother, thecotyledons of the seedlings were of a pale-yellowish, aurea, color or aurea
with streaks of pale green (chlorina). In the reciprocal cross, S X D, thecotyledons were of a healthy green, approaching the intensity of color of
those of the parents which made distinguishing them from the latter difficult. Sometimes, at nearer inspection, streaks of dark green on somewhat lighter background could be observed.
3. With the appearance of the first leaf the differences temporarily
vanished. The first leaf in both Fl's opened as aurea and when expanded
assumed a light chlorina shade producing the impression of a serious
chlorophyll deficiency.
Gradually the weak green tone of chlorina was
gaining in intensity until a darker chlorina shade was reached. The following leaves behaved in the same way so that the new growth at the
top was always before expansion aurea or very pale chlorina.
4. About two weeks later the reciprocal Fl's started to assume again
a different appearance.
While the great majority of the F l from crossD x S remained at the chlorina level, most of the plants from the reciprocal cross, S x D, started to produce more chlorophyll, turning green,
and at the same time doubling their growth rate. Soon they were becoming strong, healthy plants as vigorous as the parents, of a green somewhat
lighter when viewed together side by side. But the relatively pale colorof the new growth at the tops of the main stem and branches remained
an unmistakable indication of their hybrid origin. The plants were amply
branched and flowered generously, while in the reciprocal F l , D x S,
flowering was delayed and even later only a few flower buds were initiated.
5. This newly exhibited difference in behavior was characteristic of thegreat majority of the reciprocal Fl's. However, in either F l a few plants
were found which behaved as the majority of the reciprocal cross did,i.e. in D x S plants were found which turned green, became vigorous and
1) The designations D and S derive from a difference in the nuance of green of
the two strains; they stand, respectively, for Dusky green and Shiny green.

82

RESEARCHES CARRIED OUT IN 1959

flowered well. Likewise, some were found in S x D which remained chlorina and weak and could not be distinguished from the majority of thereciprocal cross.
6. Up to now carried out successive backcrosses of both F,'s to therespective male parent showed that a preponderantly either D- or S-genotype can function normally with either cytoplasm, its own or that of theother strain, effecting a development characteristic of the respective strain.
7. The F 2 generation presented a very similar aspect in both directionsof the cross. The appearance, in highly variable ratios, of acute growth
inhibitions and albinotic or xantha seedlings, characterizes both F 2 's.
8. Conclusion: The sensitivity relations created between the hybrid
nucleus and the plastids, directly or indirectly through the cytoplasm, arein either direction of the cross the cause of maifunction of the chiorophyii
The difference between the reciprocal crosses is
producing apparatus.
merely a difference in degree. Only the difference between the two sensitivity levels can be ascribed to a difference in cytoplasm or plastids of thetwo biotypes.
Growth inhibitions and complete chlorophyll deficiency(albinotic and almost completely chlorophyll lacking seedlings) which appear in F 2 may be attributed in first place to an intensified (as comparedwith F ,) plasma or plastid sensitivity of certain gene combinations and,partly, to gene segregation as such. The simultaneous occurrence of boththese factors complicates the analysis.
Moreover, there are indications
that a gene or gene group is involved which produces some (virus-like?)
agent exercising semi-lethal effects partly independently of the chlorophyllsituation. The behavior, under certain conditions, of the D-strain seems
to justify this supposition.
It is noteworthy that the pollen was throughout all the experiments good(in so far as the plants produced flowers).
The experiments will be reported in detail elsewhere.

56. Cytological studies in the genus Polygonum I.
Chromosome numbers in the genus Polygonum and related genera(By Yukio DOIDA)In addition to the study of pollen grain formation and pollen morphology,
the chromosome numbers in Polygonum and allied genera were examined.
The results obtained are summarised in Table 1. The following points
are noteworthy.
1) A new basic chromosome number, b=12, was found in at least 3
species of the genus; hitherto only b=10, 11, and 17 have been reported
by other authors.

RESEARCHES CARRIED OUT IN 1959
Table 1.

83

Chromosome number of the Polygonum species examined
..

.. _

----_._-~=-

Species nameI

Muehlenbeckia arisanensis Hay.
Polygonum hydropiper Linn. var.
maximowiczii (Regel) Makino
P. nipponense Makino
P. weyrichii Fr. Schm. var.
alpinum Maxim.
P. yokusaianum Makino
P. blumei Maisn.
P. persica1'ia Linn.
P. tinctm'ium Aiton.
P. japonicum Meisn.
P. thunbergii Sieb. et ZUCCo
P. sieboldi Meisn.
P. aviculare Linn.
P. nodosum Pers.
P. orientale Linn.
P. multifiorum Thunb.
P. cuspidatum Sieb. et Zucco (+)..._.._ - - ------=---~==~

p(~)C I ~~~t(~i~) I ~~~~~~~~
I

20

'\

J:
2200

So:

40
40
40
40
40
20(11)
60
22
22
22
44
24
24

Su:

I

8(11) i

24
16
16

20

I

Mt. Fuji
Misima

I'

J:

44
Su:
40
Su:
44
Su:
34
Su:
34 I
L & L: 40,60
L & L: 22,44
i J:
22
22 '
Sk:
44
J:
So:
46

J:
So:

8(11)

Misima\ Misima

20
40

iTokyo*

22,24
20

\

P. perfoliatum Linn.
P. bistorta Linn.
P. tenuicaule Bisset et Moore
Fagopyrum esculentum Moench.
F. tataricum Gaertn.

\ Localities

J:
Sd:

44
46
16
16

MisimaMisimaTokyo*
MisimaMisimaMisimaMisimaMisimaMisimaNagoya**MisimaMt. Ibuki
HakoneNagoyaNagoya------~* Supplied by Koishikawa Botanic Garden, Tokyo** Supplied by Higashiyama Botanic Garden, Nagoya
The origin of the supplied plants is not known.
J: Jaretzky (1927, 1928), So: Sokolovskaya & Sokolovskaya (1938),
Su: Sugiura (1925, 1936), L & L: Love and Love (1944),
Sk: Suzuka (1950), Sd: Sokad (1939).

2) The chromosome numbers for some species were different from thosereported by ]ARETZKY (1928) and SUGIURA (1936), perhaps because of occasional
aneuploidy.

57.

Developmental studies in the genus Polygonum III(By Yukio DOIDA)A) Two other species producing only 4 pollen grains.
In a previous paper (1958) of this series, it was reported that P. persicaria

RESEARCHES CARRIED OUT IN 1959

84

produced only four pollen grains in a pollen sac. The same has beenfound in P. debile and P. nepalense, as shown in Table 1.
Table 1.

Materials

Number of pollen grains formed in a pollen sacof P. debile and P. nepalense.

Localities

No. of pollen grainsI,--------:---! Total ! ModeI
416+2m* 8 112 I Empty I
I 3+1m* I.---_._-,- - - - - - - - - -P. debile
P. nepalenseFoot of Mt.
Fuji, Susana·cho, Shizuoka
Towada·lake,
Yasumiya,
Akita
Foot of IvIr.

P. nepalense I Hakkoda,
Sugayu, Akita
Im*:.. _~.

----- - - _ .. -

10

130

4

171

4

97

4

I

1

92

1
1

26 ! 01
1

1
0

128

0

0

! 43
1

1

0
00.

82!

0

00 _ _ - - _ -

I

15

!

I

0

_._.__
I~

Dwarf pollen grain

An anther of these species consists of four pollen sacs. A cross-sectionof a very young anther shows a mass of homogeneous meristematic cells.
Following the division of the meristematic cells, the young anther becomesslightly four-lobed. In each lobe an archesporiaI cell differentiates fromthe subepidermal layer. This cell becomes the future pollen mother cell.
Without undergoing mitosis the cell proceeds through normal meiosis andfour pollen grains are formed. Aberrations in pollen grain formation arevery few (Table 1).
The morphology of the pollen grains of both species is as follows: The
pollen grains of P. debile have a reticulate sculpture and multi-germination
pores, one in compartment of the mesh, representing the so-called" poretype". The pollen grains of P. nepalense have a similar reticulate but
pore-less structure and three germination furrows. Thus, P. nepalense
combines the reticulate structure of the pore type with the characteristicsof the furrow type. P. nepalense is the only one of this kind among
Polygonum species (Table 2).
Pollen morphology in the genus Polygonum
The external morphology of pollen grains has been used as a key
character for the study of phylogeny of some plant species. WODEHOUSE,HEDBERG, NAKAI and IKUSE classified the genus Polygonum into several
groups on the basis of their studies of pollen grains.
According to NAKAI all Polygonum species can be divided into two majorgroups, the "pore type" and the "furrow type". The grains of theformer type have an almost spherical shape and a reticulate sculpture of

B)

RESEARCHES CARRIED OUT IN 195985

the exine. Many germination-pores are found. The grains of the latter
type have spheroidal shape and spinose structure. They have three germination furrows. With regard to the number of pollen grains per sac,the present author has distinguished 7 classes, from class 1 with 4 to class
7 with 256 grains. As to the relation of NAKAI'S to the author's classification, the pore type is found in species producing a small number of pollen
grains in a pollen sac, i.e. it occurs in species belonging to classes 1, 2, 3
and to the majority of class 4. On the other hand, the "furrow type"
is observed in species producing large numbers of pollen grains. It isfound in species belonging to classes 5, 6, T and the remaining species ofclass 4, shown in Table 2.
Table 2.

Group name

Relation between NAKAI'S and theauthor's classification
Pollenmorphology

Number ofPollen grains

Classes- - - - - - _ . - - - - - .. _ - - - - - - ------------

Sub-genus FagopyrumB

32

Sub-genus Aviculare

B

32

4

Sub-genus Reynoutria
Sub-genus Pleuropterus

B
B

256

7

128

Sub-genus Bilderdykia

B

64

Sub-genus Bistorta
Sub-genus Pleuropteropyrum

B

32?
64

B

128

Sub-genus Ambryogonon

A
A

32

Sub-genus Tovara
Sub-genus Persicaria

4

5

8

6
4
2

A

Section Dissitiflorae
Section Didymocephalon

A*

4

1

Section Corynbocephalon
Section Chylocalyx

A

Section EchinocaulonA

32
4,8,16,32

1,2,3,4Section Eupersicaria

AB

4, 8,32

1,2,4

more than 1,000?

Genus Rumex
A:
A*:

4

pore type
B: furrow type
Only one species was examined (P. nepalense); it revealed characters ofboth types A. and B.

Pollen grains of the genus Rumex, Polygonaceae, belong to the" furrowtype"; they occur in much larger numbers per pollen sac than those ofPo[ygonum species.

86

RESEARCHES CARRIED OUT IN 1959

58. Biometrical analysis of matroclinal inheritance in rice 1 )(By Kan-Ichi SAKAI)

In a varietal cross of rice, matroclinal inheritance was observed for the
one hundred grain weight. On a hypothetical genetic model for the cooperation of genes and cytoplasm, theoretical expectation of mean values
for the reciprocal hybrid progenies were computed. Formulas for these
expectations are as follows:

2:- +{Cxn-1-a)- 2:- }m,

Xn=a+Table 1.

1

1Components of variation and covariation in hybrid populations.
Components of varianceor covariance

Notation- - - - - - ' - - - - - - - ' - - - - - - - - - - - -------

Parental varieties andreciprocal F 1 hybrids

I

V",o' V lIo ' V",o'

V 1I1
I

1 (
,1
2
2- D 1-m)-+4 H(l-m) +E1-Within-line variance in F s ! V"'sIWni> VlIsIWnl I

4-1 D(1-m)2+ g1 II(1-m)2+E 1

Variance in F 2

V"'2'

V 1I2

!

,1
-i1 D (1 -m)"'( l+m)-+
16 H(l-m)-

Between-line variance in F s

0(1+2ml+E2
1'
1;rD(1-m)2
L3 +4m(+ 2 m)

Variance in F s bulks

J+ 16- H
1(1-m)2[3+4m(1 +m)]+ E 1
Within-line variance in F.

11
8 D(l-ml+ 16 H(1-ml+E 1

Variance in two backcrosses between F 1 and P

i1 D(1- m )2+ Z1 H(1-m)2+2E 1

Covariance between F 2
and F sCovariance between F s
and F.

1

1i D(1-m)2(1+m)+SH(1- m l(1+2m)
1
4 D(l +m)(l-mli 3+2m(l-'-m)}
1+32 H(1-mli3+8m(1+m+m 2)}* Different backrosses are divided into two groups, X and Y.

X is a group of
backcrosses where Xo is included in the cross. Y is another group where Yo
is included. Variance of a cross of of X group and variance of a cross of Y
group can be added to give VB(XI+ VB(Y).

1)
The present study was conducted in the Department of Agriculture of Ceylonand details will be published in "Heredity" (England) as a joint work with Dr.
M. F. Chandraratna.

87

RESEARCHES CARRIED OUT IN 1959

Yn=a+

2:---;:- +{ (Yn-l-a)- 2:- }m,1

where Xn or Yn is the mean value for the n-th generation of a crossxo( -¥-) x Yo( ~) or Yo( -¥-) x xo( ~). The letters, a, h, and m stand for midparental
value, dominance effect of genes, and cytoplasmic effect, respectively.
These formulas can be rewritten asn n
Xn=mnxo+ h(l=!i1 ) (1-2 m )2n- 1 (I-2m)
h(l-m) (1-2 nm n)
Yn=mnyo+-' 2n- 1 (I-2m),

if a is O.
Components of variation in different hybrid populations have been worked
out and are presented in Table 1. For explanation of the notations D, H,
E, and E2, refer to Mather (1949): Biometrical Genetics.
Genetic parameters estimated in the investigation are as follows:
Effect of cytoplasm, m=0.3177±0.0710
Dominance effect of genes, h = - 0.4008
Fixable genetic variance, D=0.0404±0.0392
Unfixable genetic variance, H=0.2879±0.1383
Environmental variance, E , =0.0091±0.0049.
Observed values and theoretical expectations computed on the basis ofestimations given above are shown in Table 2.
Table 2. Comparison of observed and expected values of average one hundred
grain weight and variance or covariance of the same characterin different hybrid populations of a reciprocal cross.

I

I_..- - _ ..

Xo
Yo
:Cl

y,
X2
Y2
X3

~

_M_e_a~_1_0_0_g_r_ai~_~_eigh_t_(_g_)_
Observed
1. 78
3.02
2.02
2.47
2.39
2.48
2.37

Variance or covarianceJ

Expecte~V o and V ,V2

2.07362.46752.30362.42882.4451

V31WnJ

V31lJnJ
V41WnJW 2/ 3*

Obse:_v_e_d

E_x_p_ec_ted

0.0072
0.05620.02470.04150.02600.03320.0091
0.05200.03050.03900.01980.0398*J Vi stands for variance of i-th generation and Wi/} for covariance between
i-th and j-th generations.

Heritability of one hundred grain weight is 0.4009 and the number ofeffective factors for the same character is estimated at about 10.

88

RESEARCHES CARRIED OUT IN 1959

59.

Effect of matroclinal inheritance on the genetical variation
in a hybrid population of autogamous plants.
(By Shin-ya IYAMA)

Matroclinal inheritance of quantitative characters of autogamous plants
was reported on seed weight of rice by Sakai (Ann. Rep., No. 10, pp. 86)
and the variances and covariances in early generations were computed.
The following model is proposed for matroclinal inheritance.
Pn=(l-m) Gn+mPn- 1 ,where P n and Gn stand for the genetically determined phenotypic value
and genotypic value of an individual in the n-th generation, respectively,
and m stands for the proportion of the cytoplasmic effect in the whole
hereditary effect. m may take any value between zero and unity. For apair of genes A and a, assuming the values of three genotypes AA, Aa
and aa to be d, h, and -d, respectively, the mean value of the Fn progeny
population of AA x aa or aa x AA crosses of autogamous plants may be
written as follows,±mnd+_(l-m) (1-2 n m n ) h
2n - 1 (I-2m)

where ± sign means positive for AA x aa, and negative for aa x AA.
On the basis of the above assumption, general formulae for obtaining
variances and covariances in various generations of hybrid progeny havebeen worked out. The details will be reported on another opportunity.
According to the result of this study, the cytoplasmic effect reduces the
additive genetic variance more and more as the m value increases. The
effect of cytoplasm almost disappears in a few generations when m is small
but remains still effective in the later generations when m approaches 1.
Proportion if genetic variance to total phenotypic variance as well as
parent-offspring correlation coefficients were computed for various valuesof m. Both values become smaller, as the cytoplasmic effect becomeslarger. Genetic correlation, however, becomes larger as the cytoplasmic effectincreases.

60.

Biometric-genetical study of seed size charactersin cultivated varieties of ricel)(By Kan-Ichi SAKAI and M. E. R. PINTO)

Six F 2 populations, six varieties of pure-line selection, five local varieties,
and eight varieties of hybrid origin were investigated in Ceylon for the11This study was conducted in the Division of Botany, Department of Agriculture,
Ceylon.

RESEARCHES CARRIED OUT IN 1959

89

genetic variability of seed size characters. A number of plants wereselected at random from each variety or hybrid population, and ten or
twenty seeds collected at random from each plant of each variety were
investigated for seed length and seed width. Genetic variance and covariance together with environmental variance and covariance were estimated, and heritability and genetic and environmental correlations werecomputed. The frequency distribution of varieties and hybrid populations
with regard to average heritability of both characters is presented inTable 1.
Table 1.

Frequency distribution of F 2 hybrid populations and varietiesof different origin in respect to average heritability
value for seed length and seed width.
Heritability
Numberof items 100.1

0.1- 0.20.2 0.3

0.3- 0.40.4 0.5

0.50.6---~--~

F 2 population
Local variety
Pureline selection
Variety of hybrid
origin

6
5
6

1*
3

8

1

1

1
1

1

3
2

2

1

1

2

2

_

0.60.7

Mean~----_

2
i

...

_ _
•..

..

_-

0.59130.45030.15090.3365.._.. _ - -* An exceptionaIly low heritability value of one F, population is not included in
the computation of the mean value.

Mean values in the last column of Table 1 show that F, populations arehighest in heritability, local varieties coming next, followed by varieties
of hybrid origin, while varieties of pure-line selection are lowest. Thisis quite in agreement with our expectation. It should be noticed that
Table 2. Frequency distribution of varieties of different origin and F 2
hybrid populations regarding genetic correlation between
seed length and seed width.
Genetic correlation

I

Numberof items

Fe population
Local variety
Pureline variety
Variety of hybrid
origin

6
5
6
2

'I

-0.65- -0.45- -0.25- -0.05-0.46 -0.26 -0.06
0.15
2
3
1

3
1

0.16- 0.360.35 0.55

0.560.75

1
1
1

1
3

1

1

RESEARCHES CARRIED OUT IN 1959

90

varieties of hybrid orgin are very variable with respect to heritability.
It is of interest to see in Table 2 that genetic correlation between seedwidth is very low or almost none in F 2 populations while it is high in apositive or a negative direction in more or less established varieties.
Needless to say, genetic correlation would be the result of three causes:
genetic linkage, pleiotropic effect of genes, and effect of selection. Table
2 suggests that the observed genetic correlation between length and widthof seed in rice varieties is not genic but a result of natural or artificial,though probably not conscious, selection.

61.

Analyses of strains of Drosophila melanogaster selected
for abdominal bristles(By Yukio YAMADA)At the 26th generation of selection genetic analyses of variability were
undertaken in six strains of D. melanogaster selected for high and low
number of abdominal bristles. In each line 6 flies out of 30 were selectedfrom each sex. Two strains for high (HI and H2) and two for low (Land L2) bristle numbers underwent selection, and the remaining two served
as unselected controls (CI and C 2 ), all six having the same effecti"ve population size. Results so far obtained are summarized as follows:
1. Fertility:-Mean fertility, based on the number of successful matings
per 50 randomly paired matings for each line, was 69.0%, 76.5%, and86.5% for the pair of strains comprising H, L, and C, respectively, andhence it was in general lower in the selected lines than in the unselected
controls. However, the same tendency of phenodeviants to have lower
fitness than the optimaIs was not observed on a within line basis in either
selected and unselected groups.
2. Variability:-Phenotypic variability in terms of the coefficient ofvariation was substantially at the same level in all 6 strains, ranging
from 6.0% to 7.5% for abdominal bristles and 11.0% to 13.5% for sternopleurals. There was no definite evidence that the phenotypic variations
have been decreased or increased by artificial selection for 25 generations.
Heritabilities were estimated by intra-class correlation and midparentoffspring regression. The estimates obtained by the former method wererather erratic and inconsistent, but they were estimated by the regression
to be roughly 18%, 15%, and 15%, respectively, for H, C, and L lines.
Reduction of heritability of the character selected might be ascribable tothe small effective size of population in each generation.
3. Developmental stability in selected lines and their crosses: -Differencesof bristle counts of right and left sides of sternopleura were used for

91

RESEARCHES CARRIED OUT IN 1959

measuring stability in development. The difference of variances between
6 parental lines and their F,'s did not approach a statistically significant
level, though the variance in H , was the largest and that in L 2 was thesmallest. This fact does not support the general idea that stability is lostby long-term selection and that stability of crosses is superior to that ofparental lines, probably because the selected lines still preserve a great
deal of heterozygosity.
4. Genetic differences between lines: -Genetic differences between lines
were analysed from diallel crosses and some of the results are given in
Tables 1 and 2.
Analysis of variance for genetic effects from diallel crossing~

Table 1.

I
I--

Sourcesd.£.

Abdominals
I--------~~.<?.<?

Additivegenetic
Non-additive
genetic
Reciprocal
effectError

5I__._._---

Sternopleurals~-----_._-----(2)~.<?.<?

~~

44.9232**

28.2309**1.5004**

2.2165**0.44270.47080.13890.1964

1.6980**0.3934

0.5318**0.5429**0.45870.33730.09770.1210915----_.

Mean squares

I

360

!

** Significant at 1 % level.
Table 2.

Additive genetic effects assigned to each line assuming
the population mean to be zeroI

Strains

I

Abdominals

I----~iI

Hl
H2
C,
C2
L,
L2

I

I
I

I

2.92
2.48
-0.36
0.09
-2.63
-2.51

Sternopleurals~~.<?.<?

~~

2.19
2.11
-0.21
-0.39
-1.84
-2.22

0.49
0.13
0.41
-0.23
-1.33
-0.67

0.60
0.14
0.41
-0.20
-0.06
-0.80

As the tables show, the differences between H, C, and L are attributable
mostly to the additive genetic effects, though some variance ascribable toreciprocal effect are highly significant. Means of the 6 lines and their

92

RESEARCHES CARRIED OUT IN 1959

crosses are presented in Fig. 1. It is very interesting to see that themeans of crosses between the two H lines and between the two L lines
regressed somewhat to the overall population mean. This tendency seems
to be general in crossings between lines selected for a character under
additive genetic control.
45

12.'

43

12.2

a
!WI

IlxJt
!!xC

T

""
!!xC

IlxC

T

IlxC

41

11.8
I!xl
I!xL

Hr.L
2"---

I!xl
Cx<:

C

39

11.4

T

xL

exT.

T
37

1l.O

35

10.6

33

:10.)

exL

Cxl.

;>

Abdominals
Fig. 1.

62.

Sternopleurals

Means of six lines and their crosses.

Heritability analysis of body weight in purebreds andtheir crossbreds in the domestic fowl.
(By Takatada KAWAHARA)

Intra- and inter-breed matings involving White Leghorns (WL) and Barred
Plymouth Rocks (BPR) were practiced. Average coefficients of inbreeding
were 3.4% and 9.2%, respectively, in WL and BPR. Data were collectedfrom 762 purebred and 586 crossbred females originated from 16 sires and
141 dams in WL and 16 sires and 119 dams in BPR. Mating system weresuch that the purebreds and their crossbreds were half-sisters, thus comparisons were possible for groups of pure and crossbred types. Measurements of body weight were taken at 4, 8, 12, 18 weeks, at laying of thefirst egg, and at about 48 weeks of age.
Heritability estimates based on variance components for body weight atvarious ages for four groups are shown in Table 1. They are 0.675,
0.637, 0.445, and 0.637, for WL, BPR, WL-9- xBPR~ and its reciprocal,
respectively. There is some discrepancy between heritabilities as well as
between genetic coefficients of variation estimated from sire and dam components in crossbreds at various stages, especially in the period from the
4th to the 12th week. These results suggest that dominance and/or epistasis

RESEARCHES CARRIED OUT IN 1959

93

affect the growth rate in the reciprocal P, hybrids.
Table 1.

Heritability estimates of body weight in purebreds and theirreciprocal F , hybrids at various stages of growth.
Heritabilities____,:~w-:-ek-s-lw;ek~·

WL
BPR
WLif.xBPR6BPRif.x WL6-

0.96
0.65
0.21
0.53

WL
BPR
WLif.xBPR6BPRif.xWL6-

0.92
0.82
0.80
0.82

WL
BPR
WLif.xBPR6BPRif.xWL6-

0.94
0.74
0.51
0.68

!
I

w ;;ks J:~--

Age at1stegg

Adults

0.97
0.59
0.07
0.06

0.68
0.88
0.01
0.04

0.94
0.61
0.44
0.24

0.75
0.79
0.29
0.26

0.99
0.19
0.14
0.45

0.87
0.47
0.80
1.23

0.36
0.85
1.23
0.68

0.06
0.35
0.38
0.82

0.45
0.49
0.55
1.24

0.14
0.95
0.52
1.26

0.92
0.53
0.44
0.65

0.52
0.86
0.62
0.36

0.50
0.48
0.41
0.54

0.60
0.64
0.42
0.74

0.57
0.57
0.33
0.85

I

;; 8

--WL
--BPR

~

-._- BPR'IXWLt

E--WlYxBPR1>

7

S
~ 6
~
LL

o

....

..

."... / - /

.--;

5

4

z

~ 3
c;:

tb 2

o

u

_

o'---:-~-----

40..

Sw..

12w..

18...

AT hn:
EGG

AWLT
(AGEl

Fig. 1. Genetic coefficients of variation of bodyweight in purebreds and their crossbreds atvarious stages. S=100",s!x; D=lOOeJa!X.

63.

Viability and susceptibility to a visceral form leucosis in two
purebreds and their reciprocal crossbredsin the domestic fowl.
(By Takatada KAWAHARA)

Within-breed and between-breed matings were performed using 16 siresand 141 dams of White Leghorns (WL), and 16 sires and 119 dams of

...

<.0

Table 1.

Mortality of various type,; during two periods of growth .
. _----_ .. _ - - - -Maturing pE riOd------I--

Growing period

De,' bi~d'[ --.._.---

breed orcross~,-------_._--

No. Dead birds
tested _ _ - - - -

,2

...

1

29**-~-BPRI

8.3

'

1

BPRif-xWL~

II

I'

F

WL---l. 71 ~..

-BPR II(WLif-XBPR~)1

508 I 20

3.9

i40**
"-.

BPRif-xWL~

**:

*.

Significant at the 1 % level.
Significant z,t the 5;; level.

/

15.83**

"-./

FJ

I 93
-i.

I

6")3*
"-.

/
0.77

"-./

FJ

t:r1

1{J
:::0

1~2.88** I

32.41°.16'

2.05
3·10
I
18. 8 WLSf: X-4
BPRif- X
BPR~'
WL~
26.4

I

16-

o

::r:
t:r1

Ul

o

;:»
:::0
:::0

tTl

t:i

o

c:::

WL-9. 29**-BPRI
18.3

:::0

;:»

20.1 \WL-l1.80**---:7BPR

1

1

X2

1

WL--1.71--BPR 43 11.8 I_n
I ~ 13.75** I~ 2.48/ I I
253 29 11.5 7.14*~ 6.83** 53 20.9 11 6 '><: ,_ 0.03 82
I
2.25
I
9.91'''' I
nm
9 3.2 .WL¥- X ('\ ('\" BPRif-x 44 15.6 I'
96-BPR'fl X 53
I
23'
~'I
IBBR~~'
WL~
227 1149 21.5
60
30

362

BPR

,2

No. 9j-------~~

WI,-_._--_._----~-_.

IDead bi:rds

No. %No. ~%

Both period- - - - - , .-.

"cl
1? .

I 1.3 2...2

Z
.....

~

<.0

RESEARCHES CARRIED OUT IN 1959

95

Barred Plymouth Rocks (BPR). Mating systems were such that the
purebreds and the crossbreds were half-sisters. A mortality analysis ofdata given in a previous report (KAWAHARA, 1959), excluding the T2 group(KAWAHARA, 1957) and accidentally dead birds, was undertaken.
Data werecollected from 615 purebred and 508 crossbred females. The chicks weremaintained without any conscious culling up to 120 days after they laid
their first egg. Mortality was recorded for two periods, i.e. growing
period (up to the age of 18 weeks) and maturing period (from 18 weeks
to 120 days after the first egg) in this experiment. A summary of total
mortality data with the significance test based on Chi-square is given in
Table 1. The diseases observed at death were classified according to theten general types shown in Table 2. The results of statistical analysis of
the data are as follows: 1) Total mortality in the growing period ofcrossbred chicks was 5.7% less than that of purebreds, and this difference
was statistically significant at the 1% level. Total mortality of BPR was
3.2% greater than that of WL (11.5% vs. 8.3%), and mortality of crossbreds of BPRSi2- xWL'(1) was 1.7% greater than that of its reciprocal(4.9% vs. 3.2%); these differences were not statistically significant. 2)
Table 2. General categories of diseases observed at the death of purebreds
and their crossbreds up to 120 days after first egg. All percentage
are based upon the number of hatched chicks
at the start of the experiment.

WLSi2-xBPR'(1) I BPRSi2-xWL'(1)Breed or cross

WL

BPR

No. tested
Cause of--~
death
-~

362

253

281

227

2.49

9.88

6.41

11.01

2.21

3.16

2.14

0.44

2.21

2.37
0.79
0.40
1.19

o

0.44
0.44

o

o
o

o

o

o

1.58
1.98

1.08
1.08

3.08

7.73

11.07

7.47

11.01

20.1

32.4

18.8

26.4

Leucosis
Coccidiosis andparasites
Nutritional
Nervous
Circulatory
Respiratory
Eliminatory
Digestive
Reproductive
Unaccountable casesand compound
diseases
Total

1.38
0.28
0.28
0.28
1.10
2.21

0.36
0.36

o

96

RESEARCHES CARRIED OUT IN 1959

Total mortality of corssbred chicks in the maturing period is greater thanthat of purebeds (WL=11.8%, BPR=20.9%, WLif- xBPR~=15.6% andBPRif- x WL~ =21.5%), but it was only slightly hightly than in thematernal purebred half-sister group. The difference between purebreds
and maternal crossbreds in total mortality were statistically non-significant,however. 3) It is obvious that the main cause of difference in total mortality between the growing and the maturing periods was loss from visceralform leucosis. Table 2 shows also that a larger part of the difference inmortality between the various groups was due to leucosis. Deaths incrossbreds from leucosis were actually higher than in their purebredmaternal half-sisters (WL=2.5%, BPR=9.9%, WLif- xBPR~=6.4% andBPRif- xWL~ =11.0%). Mortality from this disease in crossbreds in thiscase was as high as 8.5% while is was 5.5% in purebreds and the difference
approached the significance level (x 2 =3.79, .10>P>.05). The difference of
6.2% in mortality between maternal groups=4.2% vs. BPR maternal groups=10.4%) was statistically significant at the 1% level.
Resistance and susceptibility to visceral form leucosis are controlled, inlarger part, by maternal factors and genotypes.
64.

A formula giving the effective population number basedon the sampling variance in gene frequency(By Motoo KIMURA)

The concept of the effective population number was first introduced into
population genetics by WRIGHT. Later, CROW propoed to define it fromthree different angles, i.e. (i) inbreeding effect, (ii) random extinction ofalleles and (iii) sampling variance in gene frequency.
In an attempt to derive CROW'S formula for the variance effective number(based on (iii)), I have obtained a slightly different result from that of
CROW: Let x be the frequency of an allele A in the collection of gametes
to form the (t-1)th generation and let x' be the similar frequency for the
next generation (t-th generation), then the variance effective number of
the t-th generation may be defined by
Ne!~jx(l-x)

2E{(x' -X)2} ,where E means "taking expectation of " . The new result obtained is asfollows:
1'1
2NltlNels) - - - - c , - - - - - - - (1)~k (l+ft-l)+(l-ft-l) ,where Nit) is the actual number of individuals in the t-th generation,

RESEARCHES CARRIED OUT IN 1959

97/t-, is the coefficient of inbreeding and k is the number of offspring per
individual of the (t-1) generation such that_N(t-lJk= :2 kijNit-ll
i=l
N lt - lJ_:2 (ki-k)2

sk

i~TIt_')_l {~~~~1 }11i .

This should be compared with CROW'S formula (CROW 1954) which may be
written, in the present terminology, as
It)

2NIt)

Ne(I)J=----:c-------2(2)-(1H1 +/t-,)+(l-/t-,)k
The difference between the above two formulae is that Sf appears in formula (1) while the variance 11k appears in formula (2).
It may be noted that the difference becomes negligible when the numberof individuals NIH) becomes large.
(cf. KIMURA, M. On some formulas giving the effective size of a population (in Japanese). Kwagaku Vol. 29)

65.

Some calculations on the mutational load.
(By Motoo KIMURA)

The amount of harm done to a population by mutation may be measured
by the mutational load which refers to the relative amount by which theaverage fitness of the population is decreased by the maintenance ofdeleterious genes resulting from the balance between mutation and selection. The purpose of the present report is to show the results of calculationon the mutational load under various conditions. We will designate by A 2
the mutant gene produced in each generation from its wild type allele A,
at the constant rate of J1.. We assume that generations do not overlap anddesignate the selective values of three genotypes A,A" A,A 2 and A 2A 2
as 1, 1-hs and l-s respectively (l;:o:s;:o:O, l;:O:h;:O:O).
1. 1l1utational load under random mating
For a very large random mating population, the mutational load atequilibrium may be given by

im=J1.{1-0+VO(2+0) },where

O=sh 2 j{2J1.(1-2h)}.
In figure 1, values of im are plotted against the values of h over the rangeof 1O-42h21, assuming s=l and J1.=10 5 •RESEARCHES CARRIED OUT IN 1959

98
x)'-

2.0
1.9
1.8

1.7

L 1.6
1.5
1.4

1.3

1.2
1.1

1.0, 0- 4

2S

4 5 67 80

10

-3

2

S

456788'0_ 22S456789

h.

10

-,

2

3456789.

Fig. 1.

It may be seen from this figure that, if the degree of dominance of themutant gene (h) is over 1%, the mutational load is approximately equal
to twice the mutation rate, i.e.

Lm=2/J,
with good approximation.

II.

Effect 01 inbreeding

If the population is not entirely panmictic but is slightly inbred in sucha way that it keeps a constant but low level of inbreeding in each generation, then the mutational load at equilibrium becomesL,n(f)=/J{1-(}1-¢+V((}1+¢)2+2(}1,where(}1=_h2s(1~.Q
2p(1-2h) ,Ihs
2p(1-2h)and 1 denotes the inbreeding coefficient of the population as a whole.
¢=In
Fig. 2 values of Ln(f) are plotted with a solid line against the values oflover the range of 1O-4~/~1O-1, assuming s=l, p=10- 5 and h=O.02.
On the other hand the broken line in Fig. 2 represents the mutationalload which appears in the next generation of a panmictic population when
it suddenly undergoes inbreeding of a given amount I. In this case theequation describing the load is
Lo(+ f)=Lo+Dol,
where
Do=s(1-2h) xl:h,in which Xl and X2 are respectively the equilibrium frequency of A l andA2 in the panmictic population:

99

RESEARCHES CARRIED OUT IN 1959

N~CO.

,=,. h=O.02. j'=IO-5

,,
I

t(n
-------

,
,,

,
,,
,,
I,
,
,,

L.(+f)

I

4.0

,I
,,,
,
,,
,
,,,
,
,,

3.5

I

3.0

I

I

I

/

,,

I

I

,I

,,"

L

,-

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

.'"

2.0F-_~;;;;===~--------",

1.5

4

•

"0.'

f

•

Fig. 2.

• "0"1

4, "0. 'RESEARCHES CARRIED OUT IN 1959

100

66.

On the substitutional load of a population(By Motoo KIMURA)

The substitutional load of a population, originally termed "the cost ofnatural selection" by HALDANE (1957), is the decrease of Darwinian fitness
brought obout through the process of substituting for one gene (A l ) its allelic form (A2) which is more fitted to the new environment. The amountof this decrease may be expressed in terms of loss in the geometric growthrate of the population and we may call it the substitutional load measured
in Malthusian parameters.
For a population consisting of haplont organisms, the substitutional load
which is expected if the substitution proceeds at the rate of one gene per
generation is given by

no

100

L !O
80
7ll

so

sor-----;:~.:a::.I::o·i'1 - - - -

..

40

It

. ..

I:t=::=:=~~~10"==:==:~~~::=~:~~;;:~~;::=~~~
..
1010"

IO-a

Ia.

Fig. 1.

...

1

RESEARCHES CARRIED OUT IN 1959

101

L=-logep,(l)where P is the initial frequency of the gene (A 2) involved in the substitution.
For a population of diploid organisms, the corresponding substitutionalload is shown to be as follows:
(2)

1{

I-h
}
L=-h IOgeP+(1-h)IOgeh+(1_2h)P,

where h is the degree of dominance in fitness of the gene (A2) which isto substitute its allele (AI). In Fig. 1, values of the substitutional load
given by (2) are plotted for several initial gene frequencies P over a wide
range of h (h=10-4~1). It may be seen from this figure that L increases
as P decreases, while it decreases as h increases.

67.

Substitutional load as a measure of gain in genetic information(By Motoo KIMURA)

In the process of adaptive evolution by natural selection, the amount ofgenetic information, as measured by the degree of improbability in genetic
organization, is constantly being increased.
The purpose of the present report is to show that the rate of accumulation of genetic information denoted by H is directly proportional to thesubstitutional load, namely

H=~"-=1.44L e bits per generation,loge 2where a bit is a commonly used unit of the amount of information equivalent to the information content of choosing an alternative, say 0 or 1,
with equal probability (0.5). The above relation may be derived from two
independent courses of reasonings:
1) If those individuals which are to be eliminated by natural selectionin the process of progressive evolution were kept alive and allowed toreproduce at the same rate as the favoured individuals, the population
number would become, after t generations,times its initial value. This means that natural selection allows an incident
to occur with probability one, which if without selection, could occur onlywith probability of
1/eLe' = e- Le ' •Thus information gained through t generations amounts to102

RESEARCHES CARRIED OUT IN 1959

-logze-Let=~ bits,
loge 2and therefore information gained per generation is

H=~=(1.442 .. ·)Le bits,
loge 2

as was to be shown.
2) Consider a population of haplont organisms. Let p be the initial frequency of an advantageous gene A. The probability that the gene A is
ultimately established in the population is 1 under natural selection, while
it is only p, if natural selection would not work and the fixation of genes
were left to the action of random genetic drift. Thus information conveyed
by natural selection through one gene substitution is

H=logzl-=-logzP bits.

p

On the other hand we have shown in the preceding report that for one
gene substitution
L=-logep.
Therefore

H=-logze-L=~L~.
loge 2

It is estimated, using the above relation, that the total amount of genetic
information accumulated since the beginning of Cambrian epoch (500 million years) may be of the order of 10 8 bits, if evolution has proceeded at
the standard rate (horotelic evolution).

68.

Genetical studies on the red and yellow eye pigments
in Drosophila(By Toshifumi TAIRA)

Single and multiple eye color mutant strains of Drosophila melanogasterwere used for this experiment. The relative amounts of eye pigments,
extracted from the head without the proboscis by means of the "double
extraction" method, were estimated by photometrical measurement. Several
kinds of pteridines contained mainly in the eye and in the testis wereseparated by paper chromatography, and their relative amounts were measured fluorometrically. The results are shown Table 1.
Drosophila eye pigments are of three kinds, namely, red, yellow, andbrown. Red and yellow pigments have been proved to be pteridine derivatives. A metabolic pathway from yellow to red was indicated by the

RESEARCHES CARRIED OUT IN 1959

103

experimental results. A metabolic relationship between several pteridines
was also indicated by the actions of various mutant genes affecting thepteridines.
Table 1.

Relative amounts of eye pigments and pteridines in several
mutant strains. (l'gjlO0 mg in wet weight)
Head

Strains

Body

I

RP*

YP

BFP

Total

YP

BFP

IXP

Total

bw
w

122.5
98.5
19.1
57.0
4.3
29.2
26.1
0
0

6.3
5.2
7.9
8.5
121.3
54.0
24.2
0
0

6.7
7.8
10.6
12.9
29.9
24.9
19.7
0
0

135.5
111.5
37.6
78.4
155.5
107.1
70.0
0
0

1.2
1.8
1.2
1.5
1.1
1.7
6.6
0
0

1.6
2.2
3.1
29.3
2.4
2.3
6.8
0
0

9.3
11.7
10.1
0
11.3
11.0
2.7
0
0

12.1
15.7
14.4
30.8
14.8
15.0
16.1
0
0

se
v; ry
v; se ryse ry
bw; rybw; se ry

4.1
21.9
1.0
0.5
0
0

111.6
7.2
67.7
97.2
0
0

27.4
10.1
18.1
32.0
0
0

143.1
39.2
86.8
129.7
0
0

1.8
1.1
1.0
1.6
0
0

2.2
27.2
27.0
27.1
0
0

13.6
0
0
0
0
0

!
-------

I

Ore-R
v

ca

ry
se
cl
Hn r - 3

--

-

v;

i

----------

*)

17.6
28.3
28.0
28.7
0
0

-_.... _---_.,"._ .... _ - - - - - - -RP: red pigments; YP: yellow pigment; BFP: blue fluorescent pteridine;
IXP: isoxanthopterin.
w( CommonPrecursor

bw ca

)
ry

Hn

r-3

se

cl

;

~ :

BP

(BP: biopterin)

Fig. 1.

Since the pteridines are located in the eye and in the testis, a striking
difference between the sexes could be detected; the total amount in themale body was about 20 times as large as that in the female body. The
genic actions of both wand bw are presumed to block the common pre-104

RESEARCHES CARRIED OUT IN 1959

cursor of pteridine. The precursor is more or less depressed by genic actions of ca, ry and Hn r - 3 • The homozygous ry fly was proved to lack
xanthine dehydrogenase activity which converts 2-amino-4-hYdroxypteridine(AHP) into isoxanthopterin, and the homozygous Hn r - 3 fly was considered
to have incomplete conversion of yellow pigment into AHP. The cl gene
inhibits considerably the transformation of yellow pigment into red pigment, but the se gene blocks it completely. From all those results, thescheme of pteridine metabolism in Drosophila appears to be as shown in
Fig. 1.

69.

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

We now wish to propose a structure for the yellow pigment occurringin the eyes of the mutant sepia of Drosophila melanugaster on the basisof the following findings. Upon shaking an aqueous solution of the pigment in air in the presence of borax, the yellow color of the solution
gradually disappeared. A greenish blue fluorescent compound in the reaction was identified as 7,8-dihydroxanthopterin (II) from its absorption spectrum and by chromatographic comparison with a synthetic specimen. The
other reaction product was lactic acid. This reaction did not occur in theabsence of oxygen. The molar ratio of oxygen consumed, and 7,8-dihydroxanthopterin and lactic acid produced in the reaction was 0.5: 1: 1. It isclear that the pigment carries a substituent in the 6-position of its pteridine skeleton, because 2-amino-4-hydroxypteridine-6-carboxylic acid is obtained from oxidation of the pigment. From this, it is concluded that thepigment is' 7,8-dihydropteridine having a lactyl group in its 6-position.
Basis on the evidence that polyhydroxy compounds form borate complexes,
the following schema is proposed.
OH

I

I

/'v N "-CO-CHOH-CH3 2H,O

N·

I

H2N-~N/'N/-H2

H
I

~

,--

RESEARCHES CARRIED OUT IN 1959

105

The yellow pigment in sodium bicarbonate solution consumed one moleof periodate to give acetaldehyde and a dihydropteridine, presumably 2amino-4-hydroxy-7, 8-dihydropteridine-6-carboxylic acid. This supports again
structure 1. Reduction of this dihydropteridine with sodium borohydride
gave a product with a spectrum characteristic of tetrahydropteridines.
Furthermore, photodecomposition of the pigment gave one mole each of 2amino-4-hydroxypteridine-6-carboxylic acid and acetaldehyde with the consumption of one mole of oxygen. This finding is again in accord with theproposed structure.
A possible biological importance of the pigment is suggested from its
recognition as a hydropteridine. It was recognized that the pigment (dihydropteridine) is reduced to tetrahYdro-form by the enzyme from chicken
liver which catalyses the reduction of dihydrofolic acid to tetrahydroderivative in the presence of TPNH. This suggests that in vivo the pigment may participate in oxidation-reduction systems.

70. Relations between anthocyanin contents and three factors which
condition the quality of flower color in pansies(By Toru ENDO)

The beauty of color depends upon three properties, namely, brilliance(luminosity, Y, in physical quantity), hue (dominant wave-length, dJ..), andsaturation (excitation purity, Pe). Flower colors of seven varieties ofpansies were calibrated with Matsuda Trichromatic Colorimeter and were
estimated by C.I.E. monochromatic specification (Y, dJ.. and Pe). Calibrations were made using circular parts (2 em in diameter) of the posterior
petals of three plants of each variety. Varieties were chosen for having
as different flower coloration as possible. The results are represented on
the chromaticity diagram (Fig. 1). It was found, as expected, that theflower color of each variety has a specific place on the chromaticy diagram.
Simultaneously, anthocyanin content of the posterior petals, which is themain component of flower coloration, was estimated spectrophotometricallyand compared with the above three properties. The anthocyanins wereextracted with 100 cc of 1% aqueous hydrochloric acid per 100 mg of dry
powdered petals.
As shown in Table 1, the increase of anthocyanin content results in
a considerable decrease of luminosity, but no relation between anthocyanin
content and excitation purity is indicated.

RESEARCHES CARRIED OUT IN 1959

106

y

500

0.5

O.~

C.3;

O. Z

0.1

X

Fig. 1. Places of flower colors on chromaticity diagram.
ME (Mont Blanc): white, RG (Rhinegold): yellow, RR (Raspberry Rose):
reddish purple, FB (Fire Beacon): yellowish red, AG (Alpenglow): deep
red, LT (Lake of Thun): purplish blue, B (Berna): deep purple.
Table 1.

Relative concentrations of anthocyanins and three factors
conditioning flower color.
_ _._..
Main
i Absorption
Opticalmaximamanthodensity
cyanidin
m/<
I
-~=-.

I'

Flower color

I

White
Yellow
Purplish red
Yellowish red I
Deep redi
Purplish blue
Deep purple

575
576
495c
598
616
555c
494c

73.6
52.8
4.9
7.0
3.6
10.7
1.4

15.3
66.0
43.3
71.3
51.3
34.0
23.3

517
520
518
551
523

0.960
0.196
0.900
0.455
2.580

cyanidincyanidincyanidindelphinidindelphinidin

RESEARCHES CARRIED OUT IN 1959

71.

107

Anthocyanin of purplish blue and deep purple
flowers of pansies(By Toru ENDO)

It was paper-chromatographically determined that the main anthocyanin,
violanin, of purplish blue and deep purple flowers was delphinidin-3, 5-Pcoumarylglucorhamnose (ENDo 1959). Another characterization was carriedout by the measurement of the absorption spectrum, in which E'4omiL!
E max . (S'OmiL) was about 8.4% when the anthocyanin was dissolved in 0.01%
methanolic hydrochloric acid. This value suggests that the hydroxyl groupat 5-position of the anthocyanin is substituted for sugar residue (HARBoRNE
1958).
Absorption maxima of violanin and crude extracts of flowers of bothstrains were almost the same in the methanolic solution. However, when
1% aqueous hydrochloric acid was used as extractant, the absorption maximum of purplish blue flower (551 mit) was shifted by about 27 mit towards
longer wave-length for violanin (524 mIl) but that of the deep purple (flower(523 mit) was not shifted. This shift was maintained in 2 N hydrochloric
acid even after the sample was heated for 30 min. at 77°C, but it disappeared after 17 hrs. when left at room temperature, and a large amountof precipitate was formed. It is assumed that the authocyanin of the purplish blue flower forms an additive complex with some part of the precipitate, most of which showed paper-chromatographically to be quercetin,
and also that the complex anthocyanin is easily dissociated in methanolic
solution from the quercetin-glycoside part but is comparatively stable inaqueous hydrochloric acid. In crosses between the two strains, a gene forthe production of the complex anthocyanin was dominant (or epistatic)over that controlling the free anthocyanin contained in the deep purple
flowers.

72.

Genes controlling flower coloration and their dosage
effect in competitive expression(By Toru ENDO)

Eight genes responsible for the production of anthocyanins and xanthophylls have been preliminarily determined. Their fuctions may be summarized as follows:
Xl and X 2 control the production of three xanthophylls, and have additive effect.
R controls the production of five anthocyanins, aCt, C3, D" Ds and D6,
contained in reddish flowers.

RESEARCHES CARRIED OUT IN 1959

108

RL is similar to R, but produces a smaller amount of the anthocyanins.
Probably a recessive allele to R.
B , and B 2 control the production of two anthocyanins, aC , and aD 2 ,contained in bluish flowers. Probably a gene complex.
B,L and B 2L are similar to B I and B2, but produces a smaller amount of
the anthocyanins. Probably recessive alleles to B I and B 2 •Accordingly, the genotypes with respect to flower colors of the varieties
can be represented as shown Table 1.
Table 1.
Flower color

Flower colors and their genotypes in pansies.
Production ofxanthophyll

Production of anthocyanin---------~White
Yellow
Purplish red
YeIIowish red
Deep redPurplish blue
Deep purple

Xl Xl Xl Xl

X2 X2 X2 X2

Xl Xl X, X,

X 2 X2X 2X2

Xl Xl Xl Xl

X2 X2 X2 X2

Xl Xl Xl ?
XIXIXI ?

X 2X2 X2 ?
X 2X 2X 2 ?

Xl Xl Xl Xl

X2 X2 X2 X2

X, x, X, Xl

X2 X2 X2 X2r r r rr r r rbl bl bl bl
bl bl bl blb2 b2 b2 b2
b2 b2 b2 b2

BILBILBIL?
BI BI BI ?

B 2LB 2 LB 2L?

RR??
RL RL??
R R??

B2 B2 B2 ?

It was observed that the production of anthocyanins was almost completely inhibited in some F I cross-combinations between cyanic and acyanic
varieties. Such phenomenon has been so far explained by the assumptionof an inhibiting gene. However, since the inhibition of the pansy anthocyanin formation is closely related to the presence of xanthophylls, thecontents of which are almost the same in yellow, yellowish red and deep
red varieties, the following hypothesis is proposed.
The anthocyanin and xanthophyll genes compete with one another for
a common substrate. When the ratio between dose of xanthophyll genes
X and that of reddish anthocyanin genes R is more than 4, only the xanthophylls are produced due to the dosage effect. In parallel, when theratio between dose of the xanthophyll genes and bluish anthocyanin genes
B is more than 1.5, only the xanthophylls are produced.
This hypothesis explains the F 2 segregations of cyanic x acyanic varieties,
as shown in Table 2, in which the expected values of segregation ratios
are calculated as follows:
X/R>4: yellow
X/R~4: red, reddish yellow or yellowish red
X/B?c:.1.5: yellow
X/B < 1.5: deep purple or purplish blue
X: Xl and X 2 , R: R or RL, B: B I and B 2 or BIL and B 2L

Table 2.

Genotypes of F I hybrids and frequency of acyanic plants in F 2 .
----=:c:==.:...------------~._------=,-.=:.-:=

Frequency of acyanic in F 2
Cross-combination

Flower colorof F I

Expec~~dl,obser;~lpI~nts

Genotype of F I assumed%

Iobtained

%

~

tIl

(fl

White X yellow

pale yellow

XI XI XI XI

X 2X 2 X2X2

100.0

I

100

100.0

I

White X purplish red

purplish red

Yellow X purplish red pale yellowish red

R

r rr

XI XI XIXI

X2 X2 X2 X2

25.0

I

14.3

R

r rr

XIXIXIXI

X 2X 2 X2X2

41.2

I,

33.3

i

I

tIl

;p

70

~
()

84

(fl

I

::r::
tIl

()

White X yellowish red pale purplish red

RL

r rr

XIXIXIXI

X 2 X2 X2X2

28.8

22.3

60

;p

Yellow x yellowish red yellow

RL

r rr

XIXIXIXI

X 2X 2 X 2X2

66.8

35.1

71

@

Whitexdeep red

deep redR

r rr

XI XI XIXI

X 2 X2 X2X2

25.0

16.7

72

Yellow X deep red

yellowR

r rr

XI XI XIXI

X 2 X 2X 2X2

66.8

70.0

20

~
~

0

0

c::

>-3

......

purplish blue
yellow turning to
Purplish blue X yellow
purplish blue
deep purple
Deep purple X white
Purplish blue X white

Deep purple X yellow

deep purple

BILB/-blb l

B 2LB 2'b 2b 2

XIXI XI XI

X2 X2 X2 X2

0.1

0.0

81

BI'BI'·blb l

B 2'B 21.b 2b 2

XI XI XI XI

X 2X 2 X2 X 2

19.1

21.2

33

B I B I blb l

B 2 B 2 b 2b 2

XI XI XI XI

X2 X2 X2 X2

0.1

0.0

80

B I B l blb l

B 2 B2 b 2b2

XIXIXIXl

X 2X 2 X2X2

19.1

15.0

60

Z

......
CD

U1

CD......
o

CD110

RESEARCHES CARRIED OUT IN 1959

73. Practical value of citbittol A as a reagent in
the examination of human taste(By Yoshito OGAWA)

Citbittol A is a bitter substance extracted by the writer (1957, 1958) fromthe fruit of Citrullus colocynthis, SCHRAD. The present note gives a few
data on the practical value of this substance as a reagent in the study ofhuman taste sensitivity. The threshold value of the taste sensitivity ofeach individual for Citbittol A was investigated in comparison with thatfor Pheny 1- thio-carbamide.
Aqueous solutions were made up at 20°C. in two concentrations of Citbittol A and Phenyl-thio-carbamide. Two thousand and twenty persons in
the age range of 15 to 18 years were tested. They were people residing
in Sapporo (Hokkaido), Hanamaki (lwate), Matumura (Niigata), Chiba

N.

"'

+

;d=ro
Tok 0

RESEARCHES CARRIED OUT IN 1959

111

(Chiba), Misima (Shizuoka), Hamada (Shimane), Kure (Hiroshima), Nakamura (Koochi), Miyazaki (Miyazaki) and Tokyo.
Only 48 persons (2.37%) showed no response even to a saturated solutionof Citbittol A, and 13 (0.64%) of these were also blind to Phenyl-thiocarbamide. The great majority, sensitive to Citbittol A, was composed of1972 persons (97.73%); of these 158 (7.84%) were nontesters to Phenylthio-carbamide. One thousand eight hundred and fourteen persons (89.89%)
were therefore normal tester to both chemicals.
Between the two classes, concerning their reaction to Phenyl-thio-carbamide, positive or negative, no significant difference was found in the variation of the threshold value of the sensitivity to Citbittol A (P=0.9-0.8).
And no significant difference (P=O.I) was recognized in the variation of
the threshold value of the sensitivity to Phenyl-thio-carbamide between
Citbittol tester- and non-tester groups.
Another point of interest was that the detected percentage of non-tester
to Citbittol A was higher in south-western than in north-eastern Japan.
Thus, Citbittol A seems to be a useful reagent in the examination ofhuman taste sensitivity.

74.

Subunits 01 HI gene in salmonella ll(By Tetsuo IINO)

It has been known that a flagellar protein (f1agellin) has complex antigenisities; that is, a f1agellin molecule has several different antigenicaly
active sites. A transduction experiment indicated that the specificity of
the antigen complex is determined as a whole by a single gene, HI inphase-l and H 2 in phase-2, respectively. Modifier genes for the antigenic
specificity have not been demonstrated.
The serotypes studied most extensively on antigenic subunits of thecomplex are those of g, ... group in phase-I, as listed in the KAUFFMANWHITE scheme.
The major components of the g,". group are I, g, m, s,t, p, q and u, each of which can mutate independently of the other. Amongthe serotypes which carry g,' .. group antigens, all combinations of two
components randomly chosen from g, m, s, p and t has been detected. The
u-antigen was detected in combination with g, m, P and t, but not with s.
The I-antigen accompanies either g or both g and t; q with g or both gand m. The presence of these antigenic combinations among the sero11
This work was supported by a research grant from the National Institute ofAllergy and Infectious Diseases (E-2872), Public Health Service, U.S.A.

RESEARCHES CARRIED OUT IN 1959112

types and the occurrence of mutations in the antigenic subunits make itpossible to devide the HI gene at least into five sections each of which ismarked by an antigenic specificity (Fig. 1).
Antigenic
Subunits

I!l

t

p

s
u

q
f

m
g

Fig. 1. A hypothetical schema on the distribution ofsubunit-antigen determinant in HI'

75.

Unequall recombination in salmonella ll(By Tetsuo IrNO)

In a transduction experiment from S. abony CDC103 (b: enx) to S. tyPhimurium TM 2 (i: 1.2), a recombinant type which expresses alternatively
the phase-1 antigens of each of the parental strains was obtained. The
frequency of i;;!.b variation in this strain is the same as that of phasevariation in TM 2. The antisera were prepared against each of theb-antigens of S. abony and the recombinant. The reciprocal absorption
experiment with these antisera indicated that the b-antigens in both strains
were identical. The i: b type clone does not produce any 1.2 type cells
when it is incubated on semisolid plates containing anti-i and -b sera as
selective agents. The gene which determines b-antigen type in the i: bclone is transduced into the H 2 locus; for example, a:b type is obtained intransduction from i: b- x a: enx. The transduction of Flal +, which is linked
to HI, from the i:b type to S. heidelberg SW1092 (Flal-·H,rH2 1.2) on semisolid
screening plates produces the motile i:1.2 type but not the motile b:1.2.
From these results it is inferred that the phase-1 duplicate type, i:b, isan unequall recombinant rather than a heterogenote. The stable Hlb gene
becomes unstable when it translocates to a position closely linked to Vh2+,
the stability controller of H 2 • The phenomenon is analogous with thevarigated type position effect in higher organisms. These experimental
results also suggest that the phylogenie differentiation of serotypic phase in
11This work was supported by a research grant from the National Institute ofAllergy and Infectious Diseases (E-2872), Public Health Service, U.S.A.

RESEARCHES CARRIED OUT IN 1959

113

Salmonella has passed the following courses: duplication of H, in a monophasic strain, translocation of the duplicated H, to a region proximal toVh2, and the mutations of antigen type specificity resulting in the production of the diphasic types.
76.

A relation of metabolism to UV induced mutation in yeast 1)(By Sayaka NAKAI)

Recently it has become increasingly evident that certain celluar meta-bolic conditions within a critical post-irradiation period affect the frequencyof mutation.
In this paper the effect on mutation frequency of controlled purinepyrimidine and amino acid syntheses during the "sensitive period" after
irradiation is reported. Using a multiple biochemical mutant of the haploidyeast strain (Saccharomyces cerevisiae) that requires adenine, uracil, arginine, phenylalanine, and isoleucine, it was possible to control idependently
the syntheses of these substances by supplementing them in the incubating medium. Reversion of isoleucine requiring cells to non-requiring ones"'{as used for measuring mutation frequency.
'Purine-pyrimidine synthesis: In order to study the effect of purinepyrimidine synthesis on mutation, adenine and uracil (50 ,ug/ml) were
supplemented, in media previously supplemented with all three aminoacids, at 20 minute intervals from 0 to 3 hours after irradiation. The
result obtained in this experiment is shown in Fig. 1. Mutation rate
showed a characteristic bimodal pattern.
When cells were supplemented with adenine and uracil immediately
after irradiation, the mutation frequency decreased markedly (to about
one fifth) in comparison with that obtained in the experiment without
supplement.
When supplementing was delayed, the mutation frequency increased andreached the first peak at 40 minutes.
A further delay caused a sharp
drop of mutation frequency. The second peak appeared at 100 minutes.
This result indicates that the role of nucleic acid synthesis in effecting
mutations periodically changes. It seems that the two peaks observedreflect the replication process of genetic materials, though it is yet unknown whether synthesis of DNA or RNA is responsible the peaks.
Amino acid synthesis:
In order study the effect of amino acid synthesis on mutation, a similar
experiment was carried out, i.e., arginine and phenylalanine (50 ,ug/ml)
1) This work was supported partly by Grant RF 57178 from the Rockefeller
Foundation

RESEARCHES CARRIED OUT IN 1959

114

were supplemented at various intervals to the incubation medium that waspreviously enriched with adenine and uracil. The result is also shown in
Fig. 1. A bimodal change of mutation rate was found in this experiment. This result seems to indicate that mainly protein synthesis interferes with mutation between the stages when nucleic acid synthesis has
the most prominent effect.
The results obtained in this experiment lead to the hypothesis that the
metabolic process necessary to establish a mutation involves, at least, thefollowing four sequential steps:

P~-~---'~---'~---'----'----'-~-~----'

o

a

u

Tur.£

u

n
m w
at/u J/VltLdia-tzm

~

m

m

(»·j?lid'iJ)

Fig. 1. Reverse mutation rate of isoleucine-requiring gene plottedagainst intervals between irradiation and nutritional supplement.
- - supplemented with adenine and uracil to medium pre-enrichedwith arginie, phenylalanine, and isoleucine.
. . .. supplemented with arginine and phenylalanine to medium preenriched with adenine, uracil, and isoleucine.

(1) Purine-pyrimidine synthesis decreases the frequency of mutation,
presumably due to the process of repairing radiation damage.
(2) Purine-pyrimidine synthesis promotes the occurrence of mutation.
(3) Amino acid synthesis promotes the occurrence of mutation.
(4) Purine-pyrimidine synthesis again promotes the occurrence of mutation.

77.

Cytological studies on the nucleus of yeast(By Yoshiaki YONEDA)

Many problems remain unsolved in yeast cytology. Two concepts havebeen proposed with respect to the nucleus of yeast; one is LINDEGREN'SRESEARCHES CARRIED OUT IN 1959

115

nuclear vacuole theory and the other is the concept proposed by GUILLIERMOND, who considered the body located outside the vacuole to be thenucleus. Various chromosome numbers have been reported for the one
species, Saccharomyces cerevisiae (for example, 1, 2, 4, and 5 or more as
haploid number). In order to clear up the situation, the following observations were made on the nuclear structure of S. cerevisiae.
1) Resting nucleus: In a yeast cell, a large central vacuole to which
is attached a small spherical body, is usually visible. This body was revealed by staining with Lugol's solution or acid fuchsin; it was also faintly
differentiated from the cytoplasm in a living cell under the phase contrast
microscope. It was revealed by the Feulgen technique that chromatin
substance was never found within the vacuole but was always located inthis spherical body. Thus, the latter may be assumed to be the nucleus.
The resting nucleus was composed of two parts, namely Feulgen-positive
substance of semilunar shape occupying one side of the nuclear cavity,
and the Feulgen-negative karyoplasm. In the nucleus stained with Giemsa,
a densely staining granule was observed in the center of the karyoplasm.
(2) Dividing nucleus: During budding, the Feulgen-positive chromatin
gradually changed into globular chromosomal bodies. Four such bodies
were observed in the haploid strain studied, which indicates that the haploid chromosome number is 4. The densely staining granule also divided
simultaneously with the division of the nucleus.
3) Nucleus in sporulating stage: The author observed the meiotic division process in a tetraploid strain stained with Giemsa. At first prophase,entangled thread-like chromosomes were observed, which became gradually shortened, though it was difficult to distinguish single chromosome.
At first metaphase, considerably shortened bivalent chromosomes, thenumber of which was probably eight, were observed. At that stage, thedensely staining granule was also visible.

78.

Nature of radiation-induced mutations detected in the silk-worm
by the method of specific loci 1)(By Yataro TAZIMA and Kimiharu ONIMARU)

According to the specific loci method, detected mutants might represent
several kinds of mutations, i. e., gene mutations, small deficiencies, orgross structural changes of chromosomes. It is therefore essential for the
calculation of the exact mutation rate to examine what kind of mutation
was obtained under a given traetment.
1)This work was supported by Grant RF57178 from the Rockefeller Foundation

116

RESEARCHES CARRIED OUT IN 1959

This method was employed in the silkworm in a number of irradiation
experiments, but the nature of the recovered mutants has never beenthoroughly exmined. Hence the present experiment was undertaken. The
result seemed to indicate that mutation reaction might differ according to
the locus tested. The research was, therefore, concentrated upon thecause of this seeming inconsistency between the reactions of the loci used.
a) Results with ch mutants
After irradiating +C" with X-rays and mating them to non-irradiatedpartners (the routine of the specific loci method), ch variants were pickedup among F, individuals. They were crossed again to non-irradiated wild
type. F2 from each cross comprised two kinds of lots, i. e., lots withwild type only and lots with both, wild and chocolate. The former were
presumed to be the products of sib-mating of heterozygotes for the induced
mutation. It was also assumed that all homozygotes might have been killedduring early stages.
Among 22 'mutant strains tested, 21 were found to carry embryoniclethals. The deficiency hypothesis may well explain the origin of thosemutants.
b) Results with pe and re mutants
The results obtained for pe and re were markedly different from thosefor ch. In F 2 of the recovered mutants crossed to wild type, not a single
lot was found to yield wild type only. It was, however, hardly possible
to assume that all mutation bearing chromosomes were eliminated.
The next step of the experiment was crossing the induced mutants
to + rei + re, in order to discard all individuals carrying non-irradiatedpe re chromosome. Thus, 20 mutant strains were tested in F 2 in homozygous condition for the mutation bearing chromosome. All of themshowed segregation of three different phenotypes yielding an unexpected
type pe as well as two expected types + and re.
The segregation of pe suggests that the mutation bearing chromosome
has pe and that the mutation +-> pe had occurred in the irradiated chromosome of their grand parent. Among 16 mutants, those associated withembryonic lethals were 13; one was a semi-lethal and the remaining 2
were viable.
From those findings it seems that different kinds of mutation reaction
exist according to the irradiated locus. However, the apparently different
results in the above two cases could be explained on the same basis byassuming that the majority of mutations detected at the three specific lociwere gene mutations from dominants to recessives and that the typeof mutation response is determined by the time relation between the
manifastation of the mutant genes and the srage of embryos which die

RESEARCHES CARRIED OUT IN 1959

117

due to associated lethals. The egg colour genes manifest themselves invery early stages of embryonic development, which may precede the timeof killing by the accompanying lethals, whereas the ch gene manifests its
action after completion of embryonic development.
Thus it seems likely that the principal event of mutation caused byradiation is gene mutation and whether it is associated with chromosomal
deficiency or not is merely incidental.

79.

X-ray induced dominant lethals from irradiation of different
stages of oogensis in the silkworm 1)(By Yataro T AZIMA and Kimiharu ONIMARU)

By irradiating successive stages of oogenesis, we found two radiation
sensitive stages in the silkworm. Sensitivity was measured in termes oflethal effects. The process from fifth larval stadium to early pupa is
hypersensitive, while the late pupal stage is moderately sensitive. The
former is characterized by a high number of embryos dying at an early
embryonic stage, while the latter is not marked by a particular stage ofthe dying embryos, but, if any, it would be the late embryonic stage.
Irradiation of females at the fifth larval stadium increased the incidenceof sterile moths and decreased the number of eggs formed even in fertile
moths. On the contrary, irradiation of femals at the late pupal stage
radiation effect was relatively slight so far as the egg production is concerned.
With the purpose of eliminating the effect of irradiated cytoplasm uponlethality, we made also observations of dying embryos from crosses between irradiated males and non-irradiated females. The incidence andstage specificity of the dying embryos were in good accord for both sexes
at the stage of highest mutability, i. e., fifth larval stadium day 6 in themale and late pupal stages in the female.
From those observations it was suggested that increased incidence ofdying embryos from eggs laid by females irradiated at a late pupal stage
is due to radiation-induced dominant lethals, while the marked increasein early dying embryos from females irradiated at the fifth larval stadium
is not attributed to dominant lethals, it is rather caused by disturbed egg
plasma formation due to irradiation.

1) This work was supported by Grant RF57I78 from the Rockefeller Foundation

118

80.

RESEARCHES CARRIED OUT IN 1959

Some observations relevant to the possible cause of radiation-induced
sterility in the male silkworm ll(By Yataro TAZIMA)An extremely sensitive stage to radiation was revealed early in the fifth
stadium of the male silkworm (TAZIMA, 1958), and experiments to investigate the possible cause underlying this observation have been carried out.
When males are irradiated at this particular stage and mated to nonirradiated females, most of the eggs laid are unpigmented and dry up at
a very early stage.
Two alternative hypotheses have been postulated to explain the phenomenon. One is sterility of the irradiated male due to lack or inefficiencyof sperms. The other is the killing of fertilized eggs during early cleavage by dominant lethaIs introduced with irradiated sperms. In the discussion of radiation-induced dominant lethality, it is essential to determine
which of the two may be the cause of these unpigmented eggs.
Examination of the genital organs of non-irradiated females mated toirradiated males showed that a fairly large number of sperms were ejaculated into the bursa copulatrix, while only very few sperms reached thereceptaculum seminis. This indicates that though spermiogenesis, fromthe morphological point of view, was carried out almost normally, most of
the sperms were abortive. Thus, in the majority of cases females matedto the irradiated males laid unpigmented eggs with a few exceptions ofnormally pigmented ones, i.e., fertilized eggs and in some cases abnormal
eggs with a few pigmented serosa cells. The latter ones were also foundwith approximately the same frequency among the eggs laid by virgin females, which indicated that those serosa cells might have arisen
from parthenogenetically developed nuclei.
This assumption was verified by double mating of irradiated males to
non-irradiated + females and to re females.
The comparison of the coloration of the serosa cells of abnormal eggs between the two crosses showedthat these eggs were the products of parthenogenesis, excluding the possibility of fertilization by irradiated sperms.
The conclusion is that a high incidence of unpigmented eggs in F, from
irradiated males is probably caused rather by inefficiency of the germ cells
than dominant lethaIs which manifest themeselves at an early cleavage
stage.

1) This work was supported by Grant RF57178 from the Rockefeller Foundation

RESEARCHES CARRIED OUT IN 1959

81.

119

Recovery of irradiated spermatogonia of the silkworm and thestage limit jor the production of functional sperms 1)(By Toshihiko SADO)

It was found that silkworm spermatogonia, especially in the later stages,
are easily killed by exposure to low X-ray doses. Normally much fewer
spermatogonia are found in the testes in early fifth stadium than in early
fourth stadium. It may be expected, therefore, that irradiation of males
at the fourth stadium affects fertility more than that given at the fifth
stadium. However, according to fertility tests, irradiation of males at
early fourth stadium produces less sterility than that applied at early fifth
stadium.
Hence the question arose whether less sterility in the former case wasdue to functional sperms produced from regenerated spermatogonia after
irradiation. In order to answer it, the present experiment was undertaken.
The results are as follows.
When male larvae were X-rayed at early fourth stadium with IOOOr of
X-rays, complete depletion of spermatogonia was observed three days after
irradiation but two days later regeneration occurred.
However the regenerated spermatogonia were unable to develop into functional sperms
by the time of the emergence of the moths. A number of primary spermatocytes at meiotic prophase, especially at synaptic and pachytene stages,
were also found in the testis in early fourth stadium and most of these cells
developed into functional sperms even after IOOOr of X-rays. Thus it may
be clear that the fertility of males irradiated in early fourth stadium with
IOOOr is not due to regenerated spermatogonia but due to spermato:.:ytes
irradiated at meiotic prophase 1. Another experiment showed that mostof these cells do not differentiate into functional sperms after ZOOOr of Xrays.
In groups irradiated at earlier stages, regenerated spermatogonia developed into spermatocytes 8-9 days after irradiation.
These cells were
histologically confirmed to differentiate into normal spermatozoa.
Fertility test showed that the recovered spermatogonia can develop into
mature normal sperms, if the males are irradiated early enough in thelife cycle, i. e., at the first or second stadium.

1)

This work was supported by Grant RF57178 from the Rockefeller Foundation

120

RESEARCHES CARRIED OUT IN 1959

82.

Duration of spermatogenesis in the silkwarm 1J(By Toshihiko SADO)In order to elucidate varying effects of irradiation on the successive
stages of spermatogenesis in the silkworm, it is important to know theonset and duration of each stage, especially when the recovery of irradiatedspermatogonia is taken into consideration.
No reports on this problem
have been published so far either for the silkworm or for other insects.
The answer could readily be obtained if in vitro culture of spermatoHowever, a crude estimate of
genic cells of silkworm were successful.
the duration of cell development for each stage might be possible by recording the time of the first appearance of cells of one stage and that of
the next one.
Table.

Approximate timing of cell development in the spermatogenesisof the silkworm
Stage of spermatogenesis
Spermatogonia
Spermatocytes
Meiotic prophase I
Metaphase I Anaphase II
Spermatids
Spermatozoa
Total spermatogenesis

Durationa few days
10-11 days
within 1 day
5-6 days
about 20 days

The testis of a newly hatched larva contained only a mass of priThree days after hatching primary and secondary
mordial germ cells.
spermatogonia differentiate but spermatocytes do not yet appear. Differentiation of the spermatogonia into spermatocytes occurs first in the
middle of the second instar, 5-6 days after hatching.
Then the third
stadium develops, and a high incidence of differentiation of spermatogonia
into spermatocytes is observed.
Most spermatocytes enter into a long
meiotic prophase I, starting in the middle of the second stadium and lasting until the fourth molting (16-17 days after hatching). In the beginningof the fifth stadium cells in metaphase I are observed for the first time.
Within a day after the onset of metaphase I spermiogenesis commencesin the testis, which shows that the time between metaphase I and the
beginning of spermiogenesis is very short. At the end of the fifth stadium
1) This work was supported by Grant RF57178 from the Rockefeller Foundation

RESEARCHES CARRIED OUT IN 1959

121

(22-23 days after hatching), spermatoza appear in bundles in the testes.
The above description is based on the observation of the most advanced among the germ cells.
Most of the remaining cells develop about
three or more days later.
Similar observation was made with irradiated testes. As reported previously, almost complete depletion of spermatogonia was observed threedays after irradiation of male larvae with 1000r X-rays but two days later
regeneration occurred from surviving primordial germ cells. Regenerated
germ cells entered into synaptic stage 8-9 days after irradiation and 1718 days after irradiation some cells were seen in meiotic metaphase I whilesome others were in an early spermatid stage.
They developed into
mature spermatozoa 23-24 days after irradiation.
Based on these observations approximate estimation was made for theduration of each stage of spermatogenesis, as indicated in the table.

83.

Studies on mutation rate by chronic irradiation of mice ll(By Tsutomu SUGAHARA, Kiyosi TUTIKAWA and Yoshiko TAKEDA)

In order to determine the dose of radiation and the scope of experiments necessary for the calculation of recessive lethal mutation rates, apilot experiment was carried out employing three different methods. The
results were quite useful for planning large scale experiments.
Progenies of mice of a multiple recessive stock, NH strain, irradiatedchronically with 559 r of gamma-rays for three successive generations, as
described in the Annual Report of 1958, were tested for recessive lethal
mutations from three different standpoints by mating them with the CBA
wild strain and studying their F, and F 2 progenies.
(1) Haldane's method:
Recessive lethals within definite chromosome
lengths from each marker gene were detected by searching for F, parents
with abnormal segregation in F" according to the method proposed by J.
B. S. Haldane. Recessive lethal mutation rate for the total of autosomes
was calculated from the result.
(2) Litter size: The total number of recessive lethals may be calculated
from the reduction of mean litter size in F 2 mice, provided that heterozygous recessive lethals do not have any effect on the viability of F 2 embryos
and young animals. For example, one recessive lethal in either of P mice
will reduce the F, litter size by a factor of 0.056.
(3) Sex ratio: Sex-linked recessive lethals would result in the decreaseof sex ratio in F, and F 2 • The mathematical relationship between the1)

This work was supported by Grant RF57178 from the Rockefeller Foundation

122

RESEARCHES CARRIED OUT IN 1959

shift of sex ratio and recessive lethaIs was mentioned in the previous
report.
Results of the experiment and some analysis according to these threemethods are shown in Table 1.
Table 1.
Genetical method
Haldane's method
No. of mice analysed
No. of recessive lethals
Total map length swept
Mutation rate
Observed
Expected
Litter size
No. of F 2 litters analysed
At birth
Mean litter size
Variance
At weaning
Mean litter size
Variance
Difference at brith Observed
ExpectedSex ratio
No. of F 2 mice analysed at birth
At birth
At weaning
Mutation rate
Observed
Expected

IrradiatedControl26
1
34.24 cM

13064.88 cM
4.3XlO- S jrjtotal autosomes
3.3~6.6x lO- S jrjtotal autosomes

296

148

7.57
6.790

7.21
5.133

6.96
6.995

6.51
5.696
+0.36
-0.42 ~ -0.83

2240
0.53350.54491067
0.54170.53893.7xlO- 4
2.5 ~ 5.1x 10- 4

The methods of irradiation and mating applied here should result inan accumulation of recessive lethaIs induced in the gametes in postspermatogonial stages. The results obtained by HALDANE'S method and sex
ratio were qualitatively in good accordance with the expected mutation
rate based on the assumption of HALDANE (1956) or the results on litter
size assumed by CARTER (1957), but were statistically insignificant becauseof the small number of mice used. As to litter size, the difference of themeans between irradiated and control progenies were statistically notsignificant, but the variance at birth was significantly larger in the irradiated ones than in the control. The expected result in this case wasa decrease in mean litter size which could be significant even with the

RESEARCHES CARRIED OUT IN 1959

123

number of mice used here. The unexpected result on mean litter size
seems to be associated with the increased variance.
The radiation dose and number of animals sufficient to obtain significant
results by each method were estimated from these data. Haldane's method
seemed to be the best one among the three to determine recessive lethal
mutation rate. A much larger number of animals would be necessary by
the method of sex ratio. The unexpected result with regard to litter size
should be followed up.

84.

Effect of X-rays on the rate of mitosis ll(By Tosihide H. YOSIDA and Teiichiro TAKAHASHI)

In order to investigate the rate of mitosis in the Ehrlich ascites tumor,
we X-rayed tumor bearing mice. Doses of 250 r, 500 r, 750 r, and 1,000 r
were given under the following conditions: 180 kVp; 20 mA; distance of
50 cm; filter of 0.5 mm eu + 0.5 mm AI; dose rate of 104 r/min.
In the case of 250 r, the mitotic index was markedly increased immediately after irradiation, but was reduced one hour later.
8 hours after,
an increase of mitotic cells was observed again, and still 24 hours, thenumber of mitotic cells was much higher than before irradiation. 48 hours
after irradiation, it was decreased again, and at 72 hours, the original
mitotic rate was reestablished. At 500 rand 750 r, the mitotic index wasincreased immediately after irradiation, but the increase was not as striking as at 250 r; a marked reduction of mitotic cells was observed 30
minutes after irradiation.
Their number was increased again 12 hours
after irradiation, and 12 hours later the frequency of mitosis was muchhigher than in the control batch. The frequency of mitosis at 1,000 r irradiation was similar to that at 750 r. In this case, however, the increaseof mitotic cells could not be observed immediately after irradiation.
Two stages of mitotic cells were distinguished, i. e. prometa-metaphaseand anaphase, and their frequencies were observed. Both were generally
increased immediately after irradiation with lower doses.
On the basis of the above investigations, we presume that the increaseof mitotic cells observed immadiately after irradiation is due to the stimulation of mitosis by X-rays.

1)

This work was supported by Grant RF57178 from the Rockefeller Foundation

124

RESEARCHES CARRIED OUT IN 1959

85.

Chromosome breakage at metaphase and fragment formation
at anaphase after X-irradiation! I(By Tosihide H. YOSIDA and Teiichiro TAKAHASHI)

Tumor cells of the Ehrlich ascites carcinoma were irradiated with 750 r
and 250 r under the same conditions as mentioned in the preceding paper.
After irradiation by X-rays the frequencies of 1) chromosome-, 2) chromatid- and 3) incomplete chromatid-breaks were examined immediately, 30
minutes, 8, 12, 24, 48 and 72 hours after treatment.
Immediatly after
750 r irradiation the frequency of chromosome breaks was not increased,
while 30 minutes later all three types of breaks occurred with very highfrequency, especially complete and incomplete chromatid breaks. One and8 hours after irradiation, metaphase cells could not be found. At 12 hours,
however, many mitotic cells were observed. The frequency of chromosome breaks at this time was higher than at 30 minutes after exposure,while that of complete and incomplete chromatid breaks was lower thanat 30 minutes.
The frequencies of the three types of breaks gradually
decreased with the progress of time.
The number of chromosome breaks was higher than that of fragments 30
minutes after X-irradiation at 750 r, but become similar 12 hours later.
At 24 hours after irradiation the frequencies of breaks and fragments werereversed in relation to those obtained 30 minutes after irradiation.

86.

Nature of chromosome bridges after X-irradiation 2 )(By Teiichiro TAKAHASHI and Tosihide H. YOSIDA)

Frequencies of chromosome bridges at anaphase and chromosome abnormalities such as dicentric or ring chromosomes at metaphase were
examined in the Ehrlich tumor after X-irradiation. The frequency of chroTable 1.

Frequency of chromosome abnormalities and lagging chromosomes
occurring after irradiation with 750r.
Hrs. afterType------

Chromosome abnormalities
BridgesIIn<med.
after I
....

I

0.5

0
0

0
2.2
I

12

24

48

72

0.5
3.2

0.6
2.5

0.1
1.9

0
1.5

I

I

I

1) This work was supported by Grant RF57178 from the Rockefeller Foundation
2) This work was supported by Grant RF57178 from the Rockefeller Foundation.

RESEARCHES CARRIED OUT IN 1959

125

mosome abnormalities at metaphase was very low, while that of chromosome bridges was very high (Table 1).
The majority of chromosome
bridges occurred through stickiness of the chromosome matrix.
It issuggested that the majority of chromosome bridges resulted from physiological effects of X-rays on living material.
In the case of high doses the chromosome bridges continued to appear
for a considerably longer time. Althouth it has been considered that thechromosome bridges resulting from physiological effects of X-rays are atemporary phenomenon, they continued for a longer time in the case of
Ehrlich tumar cells.

87.

Relation between micronuclei and chromosome fragments
after X-irradiation!)(By Teiichiro TAKAHASHI and Tosihide H. YOSIDA)

Micronuclei originate from fragments or lagging chromosomes. The
frequency of cells with micronuclei was not as high as that of cells withlagging chromosomes (Table 1). It is interesting that the frequency of
micronuclei after irradiation with lower doses was higher than at higher
doses, while the frequency of lagging chromosomes was comparable to thedose rate. For example, 84 per cent of cells had lagging chromosomes 24hours after irradiation at 1,000 r, but at the same time only 1.4 per centhad micronuclei.
Table 1.
----Frequency of cells with lagging chromosomes andmicronuclei after 750 r irradiation.

Hrs. after

Lagging chromosomes
Micronuclei: Immed.
after

o
0.3

0.5

12

24

48

72

28.0
0.3

76.0
0.6

76.0
5.1

28.0
8.9

30.0
2.8

100 cells were counted in each sample.

The above observations indicate that many cells with lagging chromosomes degenerate soon after cell division.

1) This work was supported by Grant RF57178 from the Rockefeller Foundation.

126

RESEARCHES CARRIED OUT IN 1959

88.

Transplantation experiments with spontaneous andradiation-induced leukemias in mice ll(By Yoshinori KURITA)

The present paper deals with a comparison of spontaneous and radiation-induced leukemias in mice.
A spontaneous leukemia developed in a 10 months old AKR/.Jax female
mouse. It had the character of so-called generalized lymphatic leukemia
and could be successfully transplanted only into AKR/Jax strain.
Thistransplantable leukemia was named AKLA line.
A leukemia induced in a C57BL/6Ms male mouse by X-irradiation wascharacterized by thymic lymphosarcoma and could be successfully transplanted only into C57BL/6Ms strain.
This transplantable leukemia wasnamed BLXL-1 line.
A comparison between the two transplantable leukemias may be summarized as follows:(1) Both leukemias could be transplanted to theoriginal strain only. They had a 100% transplantability to the respective
strain of origin.
(2) The average life span in mice bearing AKLA
leukemia was 14 days, while it was 21 days for BLXL mice.
The life
span in both cases was gradually reduced in the course of transplant generations. (3) The number of white cells was the highest 12 days after
transplantation for both leukemias. Leukemic cells in both leukemia lines,
however, always had abnormal lymphatic forms, and their granules werealways negative to peroxidase reaction. After trasplantation cells showeda daily increase in number. (4) As BLXL leukemia often showed myeloid
reactions, it may belong to the so-called" stem-cell" leukemias.
Though the opinion prevails that a hematological diagnosis in radiationinduced leukemias is not reliable, the number of white cells in BLXL line
have always reached about 50 x 10 3/mm s 12 days after transplantation andgradually reduced thereafter. Though localized BLXL leukemia began in
the thymus when transplanted into intraperitoneal cavities of other mice,it later became the so-called generalized leukemia. On the contrary, AKLA
leukemia developed as the generalized from but changed into a localized
solid form when subcutaneous transplantations of the spleen of leukemic
mice into one month old or older healthy mice were made.
The above investigations indicate that in mice there are no fundamental
differences between localized and generalized, or between spontaneous andradiation-induced, leukemias.

1)

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

RESEARCHES CARRIED OUT IN 1959

89.

127

Relation of post-irradiation treatments to radiation-induced
mutation frequency in yeast ll(By Saburo NAWA and Kazuo SAITO)

A culture of Saccharomyces sp.8256 a (su. gao rna. ME. MG. adenine)
was grown at 30°C in complete medium. Following incubation, the cells
were held for five hours at 5°C and then incubated at 30°C for two hours
to obtain synchronization of cell growth.
They were removed from themedium by centrifuging and were resuspended in 0.9 per cent saline. The
turbidities of cell suspensions were then adjusted to the optical density of
0.4 (1.0 x 10' cells/ml) at 525 m,u with saline. The suspensions were irradiated by ultraviolet light, diluted into various media, and incubated for adefinite time. Then the cells were plated on complete media. The frequency of mutation was determined by observing the change of colony
color from original pink to white after three days of incubation at 30°C.
Cell division did not occur during ten hours of incubation following
irradiation.
No significant changes in the level of survival were noted
during the course of this experiment.
When the cells were held underconditions of normal metabolism, the mutation frequency increased. Potential mutations are expected to become visible by this processes. The
mutation frequency of cells held under conditions which limit normal
metabolism was lower than that of cells held under conditions favoring
normal metabolism. Cells incubated in complete media containing chloramphenicol showed a higher frequency of mutation than cells held in unsupplemented media. The same effect of chloramphenicol was observedin cells irradiated by X-rays.
In bacteria, it has become increasingly
evident that a decline of mutation frequency occurs in the cells of postirradiation chloramphenicol treatment. If a potential irradiation induced
mutation is stabilized under one metabolism and reverted under another,
the effect of chloramphenicol in our case may be to prevent the reversion.

90.

Synthesis of contractile proteins in X-irradiated embryos of
Triturus pyrrhogaster BOIE Z1(By Yoshito OGAWA)

It was found in the early developmental stages of chick (Y. Ogawa,1) This work was supported by Grant RF57178 from the Rockefeller Foundation.
2) This work was supported by Grant RF57178 from the Rockefeller Foundation.

128

RESEARCHES CARRIED OUT IN 1959

1957, 1958) as well as Triturus pyrrhogaster, BOlE (Y. OGAWA, 1958), andin regenerating limb tissues of the latter (Y. Ogawa, 1958)1) that the formation of actin in the skeletal muscle precedes that of myosin. In consideration of this finding the following question arose: Does the formationof actin induce that of myosin and does it directly cause myosin formation?
The synthesis of contractile proteins in X-irradiated embryos of Trituruspyrrhogaster, BOlE was examined by means of the serological techniqueapplied before 1) in order to give an answer to this question.
G-actin and myosin, prepared from the skeletal muscle tissue of goodsized specimens of Triturus pyrrhogaster, were injected intravenously (fourtimes with three day intervals, 100 mg. in tatal) into rabbits. The immunized rabbit sera was raised to serological specificity by resorption testwith saline extracts of liver, spleed and skin tissue of Triturus pyrrhogaster. The titer of sera was adjusted to 1: 512, and then the precipitin
reaction with saline extract of X-irradiated embryos was carried out. A
constant volume of sera and progressively decreasing amounts of antigen
were used in order to prevent a failure due to an excess of antigen. Irradiation of embryos with 50r, 200r and 50Or was made 108 hours after
fertilization.
rrad1ation

500r

20Qr

50r

120

140

160

180

200

Fig. 1. Synthesis of contractile proteins in early development of
the embryos of Triturus pyrrhogaster irradiated by x-rays 108
hours after fertilization. Abscissa: Hours after fertilization.
Ordinate: Dose of x-rays.
1)

Refer to Report No. 107 (p. 151).

RESEARCHES CARRIED OUT IN 1959

129

In normal embryos, actin and myosin first become detectable 132 and176 hours, respectively, after fertilization (Y. OGAWA, 1958). In the case ofirradiation with 50r, both, actin and myosin, were detected 156 hours after
fertilization. The time of actin and myosin formation after fertilization
was changed to 180 and 144 hours, respectively, with 200r, and to 204 and132 hours, respectively, with 500r, as shown in Fig. 1.
X-irradiation 108 hours after fertilization, therefore, markedly supresses
the synthesis of actin and promotes that of myosin in the early developmental stages. The order of actin and myosin synthesis in the irradiatedembryos at doses of 200r and 500r was opposite to that found in the nonirradiated control.
It was thus proved that during skeletal muscle differentiation in theearly embryo of Triturus pyrrhogaster, the formation of actin does not
induce that of myosin and the processes of synthesis of both proteins are
independent of each other.

91.

Heterozygous effects of induced lethals and theirpresistence in populations ll(By Chozo OSHIMA and Osamu KITAGAWA)

Male flies of an isogenic Samarkand strain of Drosophila melanogasterwere irradiated with X-rays and the induced lethal second chromosomes
were isolated by eMI method. Four hundred and twenty three chromosomes, each irradiated with 100 r, were checked and 29 lethal (6.86%)
and 15 semi-lethal chromosomes (3.55%) were obtained. Two hundredand fifty three chromosomes, each irradiated with 500 r, were tested and7 lethals (2.77%) and 17 semi-lethals (6.72%) were detected.
Twentyseven of the above lethals have been retained in balanced condition with
the Cy inversion. The identification of each lethal was examined by diallel
crosses and two lethals (1-3, I-50) were proved to be allelic.
The heterozygous Cy/l female flies of each lethal strain were matedwith male flies of the Samarkand strain and Cy and wild type offspringwere scored. The genetic background was considered to be homozygous.
The percentages of wild type flies (lethal heterozygotes) were obtained in·each strain. These crosses were replicated ten times and the overall
average percentage of lethal heterozygotes was 46.779. The Cy/ + female
flies were mated with Samarkand male flies and the ratio of Cy and wild
type offspring was obtained. On the basis of this ratio, the overall average
percentage was corrected and the value of pre-adult viability of lethal.heterozygotes was calculated to be 0.9477. The selective disadvantage of1) This work was supported by Grant RF57178 from the Rockefeller Foundation.

130

RESEARCHES CARRIED OUT IN 1959

lethal heterozygotes is estimated at about 5 percent.
At the same time, the heterozygous Cy/l female flies of each lethal
strain were mated with male flies taken from an unrelated laboratory papulation cultured in a population cage. The laboratory population had
been made up from twenty laboratory strains and has been cultured forabout six months, and its genetic constitution was presumed to be heterozygous. The overall average percentage of lethal heterozygotes was 48.224.
The relative viability was calculated by using the relative viability obtained
Table 1.

Percentage of wild type flies (lethal heterozygotes) in offspring of
the cross, CyjlxSamarkand or Cyjlxheterozygous male.
Each value is the mean of 10 replications.
LethalHomozygous crossHeterozygous cross

I
2
3
4

47.125*36.934**47.93649.13048.06147.99744.33042.821**47.67940.922**47.006*48.38847.70449.29746.669**44.252**47.90948.59749.53148.78447.99449.50449.22143.144**47.90449.61544.600**47.00947.50547.64948.78948.26848.23547.10654.690**48.97150.01347.82046.48146.156*42.914**45.598*47.65647.24846.360*46.51846.85851.08949.63450.85551.10249.57849.89848.06946.77948.224

----

5
6
7

8
9

10
11
12
13
14
15
16
17
18
19
20
21
22

23
24
25
26
27
------------------- - - - - - -Mean* Significantly deviates from 50% at 5% level.

** Significantly deviates from 50% at 1% level.

----

RESEARCHES CARRIED OUT IN 1959

131

above:0.93139jO.87895=1.05966. The selective disadvantage, noted in homozygous background, seems to have disappeared. The results of crosses areshown in Table 1.
The analysis of variance of both crosses was made and the results are
represented in Table 2.
The differences between crosses and betweenlines (lethaIs) within a cross were highly significant. The difference between lethal strains having homozygous background was also significant,but in the case of a heterozygous background, no significant difference
was detected. The deviation from 50 percent for each lethal heterozygote
was examined and eight lethals differred significantly (1% level) in the
direction of disadvantage, while two lethals differred significantly (5%level). As a whole, there was no correlation between pre-adult viabilitiesof lethal heterozygotes in homozygous and heterozygous background. Fromthese results we can say that the selective disadvantage of lethal heterozygotes in pre-adult viability is affected by their genetic background.
Table 2.
Source of variation

Analysis of variance
D.F.

M.S.

F

- - - - - - - - - - -Total
Between crosses
Between lines within cross
Error

539
1
52
486

281.897871.150325.7166

10.9617**2.7667**

Homozygous cross- - - - ----------

Total
Between lines
Between reps
Error

269
26
9
234

91.6766
18.4595
14.08476.5089
1. 3106

269
26
9
234

50.6240
173.1142
31.9584

1.58415.4169**Heterozygous crosses
Total
Between lines
Between reps
Error

Eight lethaIs ([-7, l-8, l-10, l-18, l-19, l-22, l-24, l-26) have been kept
in separate populations which were designed by pearl's method. The
initial frequency of each lethal was 50 percent and the genetic backgroundwas homozygous. The state of their persistences in the populations was
examined six months after starting. The frequencies of lethal chromosomes decreased as shown in Table 3. Their persistence seemed to depend on the degree of selective disadvantage represented in Table 2.

132

RESEARCHES CARRIED OUT IN 1959
Table 3.

Lethal7Frequencies of lethal chromosomes in laboratory populations
6 months after starting (about 16 generations).

Replicatedpopulation

No. of chromosomes tested

C
D

64
59
60
69

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

8

10

A
C
C

D

No. of lethal
chromosomes

%

1

9.38
8.47
3.33
1.45

61
58

o

0.00

1

1.72

6
5
-

_

..

-

------

2

M
8.93
2.39
0.86

18

A
C

63
74

11
6

17.46
8.11

12.79

19

B
D

67
63

4
7

5.97
11.11

8.54

22

B
C

4.29

70
69

3

24

A
B

68
70

o
o
o

26

B
C

65
70

8
10

i

~_._---

92.

0.00

2.15

0.00
0.00

0.00

12.31
14.29

13.30

Doubling dose as a common unit of measuring mutabilities
in polygenes and major genesl)(By Yukio YAMADA)

Mutabilities in polygenes and major genes are measured by different
units and they cannot be compared with each other, because of the difficulty in estimating the number of genes belonging to a polygenic system.
The present author proposes to use the doubling dose as a common unitof measuring mutability for the comparison of mutabilities in these twokinds of genes. The theoretical basis is as follows:
Assume that spontaneous and induced mutations have the same frequency
distribution in genetic effect. Now, let f(ee) be the relative frequency of amutation with effect x at a certain locus involving a polygenic trait, such that
Spontaneous genetic variance thus may be expressed as
t1~=

[+00

J_~-X)2 f(X)fldx,

where /J. is the spontaneous mutation rate per locus. If n loci are involvedin a metric character, total genetic variance spontaneously appearing per
1)

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

RESEARCHES CARRIED OUT IN 1959

133

generation is~, 11;(i) =~, ~:~-x)2f(x)~dX= VIla,
where subscript i refers to values in the i-th locus, and V lIo is the spontaneous mutation rate in terms of variance increase per generation. From
the definition of the doubling dose, genetic variance increase per locus
under doubling dose conditions is~ :~- X)2 f(x)(2~) dx=211;
And hence, the total over-all genetic variance increase involved under
doubling dose conditions is
n

2

n

2

Vlln =2::2I1 g Ci) =22:: I1 g CiJ=2Vll O
2

i=l

i=l

Accordingly, it is deduced that the doubling dose for a polygenic system
is equivalent, as an expectation, to the amount of radiation necessary to
double the number of mutants per gene per generation, as it is the caseof major gene mutations. The doubling dose of a polygenic trait is obtained by the formula,mDz=-,;,
where Dz, m, and k are the doubling dose, the spontaneous rate of genetic
variance increase per generation, and the induced rate of genetic variation
per unit dose of radiation, respectively.
93.

Doubling dose of polygenic characters ' )(By Yokio YAMADA and Osamu KITAGAWA)

Reports on spontaneous or induced mutability of polygenic characters,such as the number of bristles on the sternite and sternopleura of DroThe mutation rate
sophila, are scanty in animals as well as in plants.
of such characters can be measured by the amount of variance increment
per unit dose in case of radiation induced mutation.
This method has
been already reported by the present authors as well as other investigators.
The mutation rate in polygenes expressed in terms of variance increment, however, can not be compared with that of major genes, since theunits of measure are entirely different, variance in the former and frequency of occurrence in the latter being used. If we want to know whether
or not mutations of polygenes occur at a higher rate than those of major
1)This work was supported by Grant RF57178 from the Rockefeller Foundation.

RESEARCHES CARRIED OUT IN 1959

134

genes, a certain common unit in estimating the rates should be used.
The difficulty in deducing mutational occurrence in polygenic characterswould be avoided by the use of doubling dose, with an appropriate assumption, which would enable us to compare the mutability of polygenic
systems with that of major genes, as is shown in the preceding article.
In order to estimate the doubling dose, we must know both spontaneousand induced mutation rates in bristle numbers on the sternite and sternopleura of Drosophila. Hitherto, the former rate was dealt with in threepapers only by DURRAT and MATHER (1954), CLAYTON and ROBERTSON (1955),
and PAXMAN (1957). The estimate obtained by DURRANT and MATHER seemed
to be inflated as was pointed out by MATHER (1956), himself. The estimates
given in the two latter reports are lower and of similar magnitude. Hence
we have used tentatively the spontaneous mutation rates of those authors,
0.005 and 0.001, respectively, for the estimation of the doubling dose inabdominal and sternopleural bristle number, before our own estimates of
spontaneous mutation rate in these characters are obtained.
The experimental procedure was similar as described in the previous
report (1958). However, lower radiation doses, 500r, 1000r and 1500r wereused in the present experiment. The means and variances of bristle numbers, which were pooled for sexes and replicates, are tabulated as follows:
Or

Dose

500r

1000r

1500r

42

45

50

-----------

Genomessampled----------

Abdominals

Sternopleurals

54-------

x29.1155

28.6081

28.760928.8297

6'2

0.48030.44040.74880.6774x

14.271414.6337

14.7945

14.7100

0.40800.55840.48340.5406

6'2----- ----------------

From the results, our estimates of induced mutation rates are 8.7 x 10- 5
for abdominal bristles and 3.5 x 10- 5 per rad for sternopleurals in termsof variance increment under random mating. Thus, we obtain D,=57·5r
and D2 =28.6r, respectively, for the abdominal and sternopleural bristle
number in Drosophila. It may, therefore, be concluded that mutability inthese polygenic systems is substantially of the same magnitude as that ofmajor genes.

RESEARCHES CARRIED OUT IN 1959

94.

135

Radiation effects on germ cells of rainbow trout(By Yusiro HARADAlI, Tosio AI21, Tosio AI31 and Sohei KONDO)

Three old male and female rainbow trout were irradiated with acute
X-ray doses of 40 to 1200 r and the sperms and eggs were pressed out byhands.
The radiation effects on the germ cells were tested by artificially fertilizing non-irradiated eggs with irradiated sperms or irradiated eggs with
non-irradiated sperms.
Observations were made on the number of unfertilized eggs recovered as the ratio of the number of eggs whose embryos died before the stage of eye formation to the total number of eggs,
and on the number of post-embryonic lethals recovered as the ratio of thenumber of unhatched eggs to that of the eggs whose embryos had reached
the stage of eye formation.
I-<

o ~ 1
if)

ro

Q)

~

7-

011..<::
011""
"0

u

Q)

§
I-<
Q)

'+<

<=1
;:l

'+<

u

'5
I-<

..0

S
Q)

1

~

n

-- I

if)

0

...,

0.;..;

~

I-<

..0

8

if)

p.

Q)

ro

o>,

!

..:,

0

Or

'5

0
>,

0

8.

,-/220r

'0
Q)

V

...,

44Dr
x

~880r

mOr

p;j IOOL..:~====::;:::::=;:=:":"'_
20

Fig. 1.
Rate of unfertilized eggs.
Rate of post-embryonic lethals.
Symbol I indicates irradiation of young germ
cells and II irradiation of mature sperms(C!)) or mature ovaries (.¥-).

4C

60

Days after X-ray irradiation
Fig. 2. Recovery tests of germ
cells of male trout exposed to
X-rays in the crossing season.

( I) Relation between radiation sensitivity of male germ cells and theil
developmental stages: Acute X-rays were given to two groups of male
trouts, one so early in the hatching season that pressure on their abdomens did not release sperms and the other in the crossing season when
sperms were quite easily released by a slight touch. Later these sperms
were used to artificially fertilize non-irradiated eggs.
25% unfertilized
1)
2)
3)

Prefectural Fisheries Experimental Station, Simizu and
Simoda.
Huzi Trout Breeding Station, Inogasira.

136

RESEARCHES CARRIED OUT IN 1959

eggs were reached at about 300 r for both groups. The rate of postembryonic lethals was, however, appreciably higher for males irradiatedearly in season than for those irradiated during the crossing season. For
example, the dose of 300 r was strong enough to kill 100% of the fertilized eggs of the former while the same dose killed only 60% of thefertilized eggs of the latter.
This means that young germ cells of male
trout evaluated in terms of post-embryonic lethaIs are more radiation sensitive than mature sperms as is the case in the mouse and the silkworm.
( II ) Comparison of radiation sensitivity between male and female trout:
Radiation sensitivity of male trout in the crossing season was comparedwith that of female trout. The number of unfertilized eggs and post-embryonic lethals was scored. Fig.l shows that the female germ cells proved
to be much more resistant to radiation injury than the male cells.
(III) Recovery of X-rayed germ cells: To test the recovery of X-rayed
germ cells, we took successive batches of sperm from the same X-rayedfish at intervals after the exposure and used them to artificially fertilize
non-irradiated eggs. Fig. 2 shows that, in terms of post-embryonic lethality,
there has been no male germ cell recovery from radiation injury during
the period 3 to 66 days after X-ray exposure, although there has been aslight recovery of egg fertility.
The eggs of rainbow trout are much more resistant to X-rays than the
sperms. Young germ cells of male trout are more radiation sensitive thanmature sperms. Radiation injury of male' germ cells results in a strikingly
high frequency of dominant lethals at the post-embryonic stage.
Only
200 r irradiation of adult males is potent enough to kill 50% of embryos
during the stages between eye formation and hatching. The germ cells
do not recover from these radiation injuries.

95.

Comparison of radiation effects of {3- and i-rays
on einkorn wheat(By Seiji MATSUMURA)

Seeds of Triticum monococcum fiavescens were soaked in 32p and 131 1
solutions for 2 days before sowing, to compare the radiation effects of {3rays with those of i-rays. Radioactive solutions contained 0.15, 0.3 and0.6 mcjgr of 32p and 0.6 mc/gr of 1311. Also i-irradiation with BOCO wasapplied at the dosages of 2.5, 5 and 10 kr immediately after soaking theseeds in water for 2 days. The growth of seedlings, height of matureplants, single-spike fertility, and chromosome aberrations of treated plants

RESEARCHES CARRIED OUT IN 1959

137

in Xl and chlorophyll mutations in X 2 were compared for [3- and r-irradiations. The data are shown in Table 1.
The higher the dosage of [3- and r-rays, the more delayed were thegermination and growth of seedlings and the lower were the survival rate,height of mature plants, and fertility. The relation between the inhibitionof seedling growth and dosage of [3- and r-radiations coincides roughlywith that between the decrease of survival rate or fertility and dosage.
There was poor germination and little growth of seedlings at 10 kr r-irradiation and at 0.6 mcjgr 32p [3-irradiation, both irradiations being markedly
effective. 5 kr r-rays and 0.3 mcjgr 32p [3-rays considerably inhibited thegrowth of seedlings and reduced the survival rate and fertility. But 2.5 kr
r-irradiation was only slightly effective and the effects corresponded roughly
to those of 0.15 mcjgr [3-radiation 32p and 0.6 mcjgr 131 1 solutions. The
frequency of ears with chromosome aberrations in Xl-plants and headprogenies with chlorophyll mutations in the X 2 -generation increased generally with the increase of [3- and r-radiation dosage. Again the effects
of 0.15 mcjgr [3-radiation 32p and 0.6 mcjgr 131 1 solutions corresponded
roughly to those of 2.5 kr r-radiation.
Table 1.

DosageRadiation effects of (3- and r-rays in einkorn wheat.

Germina- Length of Survival Plant Fertil- Chromosome Chlorophylltion rate seedlings*
rateheight ity in aberrations
mutations
(%)
(em)
(%)
(%) Xl (%) in PMC (%) in X 2 (%)"--------_._---

Control
98.00
2.5kr 94.00
r-rays
5.0kr 90.00
10.0kr 50.00

8.75
7.29
2.06
0.48

54.00
48.00
20.00

95.26
98.71
96.30

89.22
71.24
51.80

0.00
2.27
64.00

0.00
1.67
10.00

(0.15 mejgr 96.00
32p
0 .3 mejgr 90.00
lO.6 mejgr 70.00

1

7.23
2.69
1.01

46.00
28.00

91.43
89.79

62.47
56.52

6.06
33.33

15.56
5.00

131 1

0.6 mejgr 98.00

7.86

48.00

93.42

77.51

2.08

6.45

1

* The seedlings were measured 3 weeks after sowing.

These findings generally confirm the experiments performed last year.
If we assume that the effects of [3-radiation are confined to the embryo,
we find by calculation that the 0.15 mcjgr 32p solution equals about 2 krad.
This, too, will account for the obtained data.

138

RESEARCHES CARRIED OUT IN 1959

96.

Radiation effects of fast and thermal neutrons on wheatA) Genetic effects of neutrons on einkorn wheat(By Seiji MATSUMURA)

Thermal neutron irradiations were conducted in the thermal column of
the Japan Atomic Energy Research Institute's Nuclear Reactor, JRR-1.
Hole No. 7 was selected to keep r-contamination as small as possible. The
thermal neutron flux was calculated to be 4.2 (V), 2.3 (IV), 1.32 (III), 0.78
(II) and 0.49 x 10 3 nUl/cm 2 ·sec (I) at the distances 4, 9, 14, 19 and 24 cm fromthe thermal column when the reactor was operated at 40 kilowatts. Dormant seeds of Triticum monococcum flavescens were treated at five different
distances (I~V) for 2 weeks (actually 990.8 kWh). The thermal neutron
integrated flux ranged from 4.4 to 37.5 x 10 12 nth/cm 2 • The data are shownin Table 1. The irradiated seeds were almost uniformly injured in each
Table 1.

Effects of thermal neutrons on einkorn wheat.

ThermalLengthneutron I Germi- Iofflux
nationseed12· (x 10nth I (%) I lings*
I
Icm 2 )
] 1 (Index)

I

Control
I
II

III
N

V

4.4
7.0
11.8
20.6
37.5

100
98
98
96
68
0

100
101.5
82.7
69.5
27.7

i

Survival
(%)

I

100
90
90
82
36

100
89.2
102.2 I 71.5
92.8 . 52.9
91.3 I 47.2
87.3
37.1

0.0
8.5
15.1
20.6
25.0

0.0
15.2
16.4
22.6

I

* The seedlings were measured 14 days after sowing.

treatment. There was no germination in (V) and about 2/3 of seeds ger
minated but about one half of the seedlings soon died in (IV). The higher
the dosage of thermal neutrons, the more delayed were germination andgrowth of seedlings and the more reduced were survival rate, height ofmature plants, and seed fertility. The frequency of chromosome aberrations and chlorophyll mutations increased with the increase of dosage,as expected.
If we assume that r-contamination for 10 12 nth/cm 2 is 430 r by Dr.
KONDO'S measurement with FRICKE'S dosimeter, the 1 r equivalent effects
produced by the thermal neutrons are calculated as follows, comparedwith the results produced last year by X- and r-irradiation at 10 and 20 kr:
for seedling growth, 1 r =2.0 x 10 9 nth/cm'

RESEARCHES CARRIED OUT IN 1959139

for seed fertility, 1 r= 1.4 x 10 9 nth/cm 2for chromosome aberration, 1 r=1.2 x 10 9 nth/cm 2for chlorophyll mutation, 1 r=2.3 X 10 8 nth/cm2
The exposure to 14 MeV neutrons obtained from (D, T) reaction was
carried out in the Biology Division, Oak Ridge National Laboratory, bycourtesy of Dr. M. L. RANDOLPH and Mr. D. L. PARRISH. Dormant seedsof T. monococcum fiavescens were fixed at 6.3 cm from the tritium target
Table 2.

Effects of fast neutrons on einkorn wheat.
I

I

DosageI

I

LengthI
Ferti- Chromo- I ChloroGermi- IofSurviPlant'some aberphyll
nation seedlings* I val i heightlity I rations in mutations
PMC's I I·n X(;Yo) I(;Yo)
( Index) " in Xl
(;Yo)
, 2
(Index)
(;Yo)
I
(;Yo)
1

- -

I

---------------;----~---:------

Control
10 kr
X-ray {
20kr
10 kr
r-ray {
20kr
0.5 krad1.0 krad1.5 krad2.0 krad2.5 krad5.0 krad7.5-20 krad
*

100
94
40
94
60
100
100
96
94
74
60
16~26

100

95 . 9

100

75.6
42.3
84.9
14.4
90.7

68.0
0
76.0
16.0
93.9

9~6

72.4
50.5
31.0
6.6

90.0
66.0
4.0
0

I

3.8

I

0

1.8~3.8

I

0

I

47.2

29.4

11.7

97.4 I 61.5
95.2
52.7
92.2
50.1
88.6 I 52.6
73.9 i 33.6
82.7
7.7

26.6
20.0
10.5
23.7
28.6

5.1
9.8
20.0

I,

1

The seedlings were measured 14 days after sowing.

and the exposure periods were varied from 7.7 min. to 306 min. The calculation of given doses is based on the first collision dose method developed by RANDOLPH who uses the analyzed elemental compositions and themeasured neutron fluxes (d. KONDO 1959, Ann. Rep. (in Japanese) No.9:
135). The dosage ranged from 0.5 to 20 krad. At the same time at
ORNL X- and r-radiations of 10 and 20 kr were used for comparison.
The effects of both radiations seemed to be rather stronger than thosefound in the same experiments last year in our institute. There was no
germination or almost none at more than 2 krad of fast neutrons. At
0.5~2 krad, survival rate, height of mature plants, and seed fertility decreased, and chromosome aberrations and chlorophyll mutations increased
with the increase of dosage.
Relative biological effectiveness (RBE) for fast neutrons, compared with

140

RESEARCHES CARRIED OUT IN 1959

x- and r-rays, is calculated as follows:for germination rate,
for seedling growth,for seed fertility,
for chromosome aberration,

97.

B)

3.5
10
11.8
8

Relation of ploidy to chromosome aberrations(By Mitsuya NEZU)

Meiotic irregularities induced by various irradiations were compared in
tetra- and hexaploid wheat. The experimental data are shown in Table 3.
Chromosome aberrations induced by thermal neutrons produced by JRR1 were mostly univalents and reciprocal translocations (® and ®). Univalents and ® were more prominent in 6x species than in 4x species.
Average number of breaks per cell increased non-linearly with the increaseof dosage, when an univalent was counted as one, ® as two, and ® asthree breaks. The number of breaks in 6x was about 3 times as high as
in 4x species. No increase of breaks was found between treatments (II)and (III) in either species. The number of breaks obtained by these treatments was comparable to that obtained by 10 kr X- or r-rays. Seed fertility decreased with the increasing dosage in both 4x and 6x. The 6x was
slightly more susceptible than the 4x in this respect.
The plants irradiated at more than 2.5 krad of fast neutrons at ORNL
did not grow. Meiotic irregularities only could be examined in plants irradiated at 2.5 krad. All chromosome configurations in 4x species were
easily analyzed. At 2.5 krad the number of breaks observed was 4.47 percell, which was about twice of as much as at X-irradiation of 20 kr. In
the 6x species, a few cells had one complicated multivalent and many
fragments which made an estimation of the number of breakages difficult.
Estimated only from clear aberrations it was 5.62 per cell. This estimation might be somewhat too low. Both X- and r-irradiations at 10 and
20 kr caused about 3 times as many breakages in the 6x species as in the
4x.
X-irradiation produced about 1.5 times as many breakages as r-irradiation at 10 and 20 kr of 4x and 6x. There was no difference between
sensitivities of the 4x and 6x species with regard to X- and r-irradiation
effect on fertility.

Table 3. Frequency of chromosome aberrations at
JRR-1 (Thermal neutrons)__-I'
Material"-"• Total n~-.-----.
D~sage 2 I(x 10 nth/cm ),I

ControlT. durumI ( 4.4)(4x)

II ( 7.0)
ill (11.8)

N (20.6)
V (37.5)
Control
I ( 4.4)
II ( 7.0)
ill (11.8)
N (20.6)

T. vulgare(6x)metaphase-! in PMC's induced by neutrons in polyploid wheats.

.___

_

of c e l l s - - - - - - .
observed
Fragments
UllIvalents

18
33
38
34
30

@

®

®

®

0·· _ _ . _ . - - - - - - - - - -No. of cells
having otheraberrations

Average no.
of breaks
per cellmonosomic 1

0.00
0.42
0.86
0.76
1.60
3.42

7
4
2

2

5

7

13
14
24
24
25

-----

Total no~-o~;d

4

12
8
2

16

14
12
17
12

1

1

0.30
0.85
2.54

2
4

20
21
27

3

2.66

1

3
9

many
fragments 4

5.16

V (37.5)
ORNL

T. durum(4x)

T. vulgarer ~.;;,,:l ~(6x)

:::d
tTl
(f)
tTl
~

p;

::r:
tTl

(f)

(")

~

:::d
:::d

til
t:l
o

c::>-jX-lO kr
X-20 kr
r-lO kr
r-20 kr
fast neutron
2.5 krad

33
31
28
27
23

2
8

12
30

2

5

4

19
18

X-lO kr

23

X-20 kr62

r-10 kr
r-20 kr
fast neutron
2.5 krad

29
14
15

1

14

21
10
30
17

7

12

17

11

11
4

1

1
2
1
2
11
5

monosomic 1

2

3

® 1
monosomic 1

7

1

many fragments 2complex 1

0.87
2.38
0.53
1.62
4.47

Z
......

~

<D

2.95
8.00
2.31
5.57
5.26

................

142

RESEARCHES CARRIED OUT IN 1959

98.

C)

Relation between ploidy and radiation effects(By Seiji MATSUMURA and Mitsuya NEZU)

Thermal neutron irradiations were conducted in the JRR-l Reactor, as
in the case of T. monococcum. Exposure to 14 MeV fast neutrons at ORNL
was simultaneously carried out with T. monococcum, 1'. durum, and T.
vulgare. The dosage ranged from 2.5 to 20 krad for T. durum and T.
vulgare. After exposure to 37.5 x 10 12 nth/cm 2 (V) and 5 krad of fast neutrons, T. monococcum did not germinate, while the seeds of T. durum and
T. vulgare germinated but most of the seedlings died at an early stage.
At 20.6 x 10 12 nth/cm 2 (IV) and 2~2.5 krad of fast neutrons most of theseedlings of T. monococcum died, while in T. durum and T. vulgare slow
growth of seedlings continued. The higher was the dosage of thermal
and fast neutrons, the more delayed were germination and growth ofseedlings and the more reduced were survival rate, height of matureplants, and seed fertility. These relationships were ascertained in T.
durum and T. vulgare, as well as in T. monococcum. In general, T.
monococcum is the most sensitive to thermal and fast neutrons and T.
durum is unexpectedly the most resistant. There is no significant difTable 4.

Comparison of the thermal neutron flux having the 1r equivalenteffects (x 109 ntb/em2) in 2x, 4x and 6x wheats.
----_.---------==:::..

-~--=---

PloidyLength ofseedlings

Seed
fertility

Chromosome
aberrations

Chlorophyllmutations

2x
4x
6x

1.6
3.6
4.9

2.6
2.7
5.1

4.1

0.73

2.4
2.0

ference between T. durum and T. vulgare. Table 4 shows the thermal
neutron flux having the lr equivalent effects calculated for 2x, 4x, 6x
Table 5.

Comparison of fast neutron RBE in 2x, 4x and 6x wheats.

PloidyGermination rate

2x
4x
6x

3.5
3.3
3.0

i
I

Length ofseedlings

Seed
fertility

10.0

11.8
20.0
10-17

5.5
5.5

i

Chromosome Chlorophyll
aberrations i mutations
8
26

10

~10

species, and comparison is given with the results obtained by 10 and
20 kr X- and r-irradiations at ORNL.

RESEARCHES CARRIED OUT IN 1959

143

RBE of fast neutrons in comparison with X- and r-rays is calculated asshown in Table 5. RBE is lower for the early stage characters (germination rate, length of seedlings) than for those of mature plants (seed fertility, chromosome aberrations), especially in polyploids.

99.

Radiosensitivity in polyPloid plants(By Taro FUJII and Seiji MATSUMURA)Radiosensitivity of plants under the same physiological conditions varies
according to the species or variety. Moreover, it is well known that radiosensitivity also varies according to the chromosome number, polyploid orheteroploid, within the same genus or species. Dry dormant seeds of Beta,
Raphanus, Astragalus, Capsicum and Citrullus and their autotetraploids
were irradiated by r-rays from a 60CO source. The behavior of autotetraploids was clear. In general, they showed a higher tolerance to radiation
than did the respective diploids. Tetraploid germination and survival rates
in all materials were higher than diploid rates.
In Triticum and Aegilops, diploid species were the most sensitive toradiation but no clear difference in sensitivity was found between allotetra- and hexaploid wheat species. Among Oryza species, O. officinalis(n=12) was the most sensitive, while O. sativa f. spontanea (n=12) was
the most resistant to radiation. Four tetraploid species, O. alata, O.
eichingeri, O. minuta and O. lati/olia (the last two surely alloploids),
showed moderate sensitivity. Nicotiana species with low chromosome
numbers (N. alata n=9, N. Langsdorffii n=9, N. longijlora n=lO) seemed to be very sensitive and the allotetraploid species (N. rustica n=24,
N. tabacum n=24) were the most resistant among the American species.
Four Australian species with different chromosome numbers, namely N.
suaveolens (n=16), N. Gossei (n=18), N. megalosiphon (n=22) and N.
rotundi/olia (n=22) showed relatively high radioresistance and their germination rates were similar to those of the tetraploid American species. On
the contrary, N. Debneyi (n=24) with the highest chromosome number
among the Australian species was the most sensitive. Furthermore, theamphidiploid N. tabacum x N. Debneyi showed high susceptibility, thoughhaving the highest chromosome number of all materials. In Nicotiana asin Oryza, no clear relation between chromosome numbers and radiosensitivity was recognized. These findings show that radiosensitivity depends
not noly on the number of genomes but also on the genome constitution,
especially in allopolyploid plants (d. FUJII and MATSUMURA 1959, Jap. Jour.
Breed. 9).

144

RESEARCHES CARRIED OUT IN 1959

100.

Radiosensitivity in vegetatively propagated plants(By Seiji MATSUMURA andTaro FUJII)Radiosensitivity is different according to the species or genus, and isalso dependent on physiological conditions; it is strongly affected by watercontent, dormancy, and metabolic activity. The determination of radiosensitivity of cuttings, tubers or bulbs of vegetatively propagated plants
is interesting to the radiobiologist. Moreover, breeding from bud or somatic mutations is widely used in these plants.
Irradiation experiments were carried out with several vegetatively propagated plants, and growth inhibition and morphological abnormalities in
the irradiated generation were observed.
Cuttings of lemon trees were subjected to X- and r-ray treatments of
1,.....,20 kr. The percentage of sprouting decreased roughly with the dosage
increase, but a promoting effect in sprouting ability was shown at low
doses of X- and r-rays. Stolons of two commercial varieties of strawberries were irradiated with X- and r-rays of 0.5,.....,4 kr. There was almost no
difference in growth between the untreated and the irradiated groups.
Only a slight inhibition was observed at a higher dosage. Bulbs of several
commercial varieties of tulips were irradiated with r-rays of 1,.....,3kr. The
percentage of flowering bulbs in the irradiated generation decreased with
the increase of dosage, while the irradiation effect was not clear with respect to sprouting rate. In all flowers of the variety Spring Field obtained from irradiated bulbs small red spots appeared over the whole surfaceof the petals and their density increased with the increase of dosage. A
red flowering variety Marshal Haig when irradiated had split petal margins, especially on the tips, the splitting increasing with the increase of
dose. Bulbs of four commercial varieties of gladiolus were irradiated with
1 to 5 kr of r-rays. Irradiation did not affect either the percentage ofsprouting and flowering plants or the average plant height.
According to our experiments, the optimum dosage of radiation forgenetical study and plant breeding from cuttings, tubers, and bulbs isabout 1/10 of that commonly applied to dormant seeds. In seed propagated plants the direction of mutation in most cases is from dominant torecessive. Therefore, detection of mutated homozygotes is observable
only in X2 or successive generations. But recessive mutation is sometimes
detectable in the Xl generation of vegetatively propagated plants, because
they are generally heterozygotes. The flower color was altered by radiation with relatively high frequency and relatively simple biochemicalchange. Thus, irradiation of shoots, tubers and bulbs may be useful as

RESEARCHES CARRIED OUT IN 1959

145

a method of plant breeding in vegetatively propagated plants (d. MATSUMURA and FUJII 1959, Rep. Kihara lnst. BioI. Res. 10).

101.

Cytological studies on X-rayed rice
(By Chen-seng HUANGII)

The purpose of this work was to find out if, during the growth of rice,a competition takes place between cells with chromosome aberrations andnormal cells. Dormant seeds of rice were irradiated with 5, 10, 20, 30 and
40 kr. Root tips and pollen mother cells of X, plants were fixed for chromosome observation. The former were stained by FEULGEN'S reaction andthe latter with aceto-carmine. Chromosome bridges at mitotic anaphase were
not clear and appeared to be caused by stickiness; therefore observations
were made mainly at metaphase. Most of the chromosome aberrations
observed at metaphase were fragments and dicentric or more complex
chromosomes, and the frequency of cells with aberrations increased linearlywith the increase of dosage .. Chromosome aberrations in pollen mother cellsof X, plants were mostly double, single and reciprocal translocations. The
frequency of translocations also increased linearly up to 20 kr but remained
constant between 20 and 30 kr. No competition between cells with translocations and those without was found in 5 kr plot, when root tip and PMC
data were compared. In 10 and 20 kr plots, about 8% of cells with translocations were eliminated during successive somatic divisions, and about
40% were eliminated in 30 kr plot. The seed fertility per pancile of Xlplants, which ranged from zero to 100 percent, was also investigated.
Three peaks-i.e., fertile, semisterile and sterile-were found in 20 and 30
kr plots, while only 2 peaks were found in 5 and 10 kr plots. The threepeaks can be explained as due to normal behavior, single and double translocations, respectively.
It is noteworthy that no somatic selection was found at as low dosage
as 5 kr. Generally, the frequency of spontaneously occurring translocationis very low, but cells with translocations may survive. Under natural
conditions, translocations of chromosomes occur in the somatic stage ofrice and result in mutations in the reproduction stage.

11

Taiwan Agricultural Research Institute, Taipei, Taiwan, China.

146

RESEARCHES CARRIED OUT IN 1959

102.

Variation in the frequency of tetraPloid cells in the courseof transplantations of the Yoshida sarcoma(By Tosihide H. YOSIDA)

The Yoshida rat sarcoma obtained from the Pharmacological ResearchInstitute of Tokyo was characterized by tumor stem cells containing 40
chromosomes and only 3.4 per cent near-tetraploid tumor cells (Figs. 1 and
2). The frequency of the tetraploid cells in this tumor has never changedin the couse of serial transplantation to W-strain rats. Surprising was thefact that about 51 per cent of the tumor cells which had developed byinoculation of a rat of B (Buffalo)-strain showed tetraploidy. The numberof tetraploid cells increased to 61.1 per cent at the next transplant generation in rats of the SH-strain. Moreover, it increased also in N- and Wstrains in the following transplant generation, while it markedly decreased

Figs. 1 and 2. Chromosomes of Yoshida sarcoma cells.
1, near-diploid cell (8=40) 2, near-tetraploid cell (28=80).

when transplanted to the LE-strain. Since the frequency of tetraploid cells
differed considerably according to the strain, rats of various strains, such
as WKA, W, N, WPY, LE, B, CW, were used for transplantation ofa tumor which had developed in the N-strain. The frequency of tetraploidcells in these tumors decreased generally to 25-35%. They decreased
gradually in the course of successive transplant generations to rats of all
strains; in the 9th to 10th transplant generations they amounted to about
10 per cent. Although a higher percentage of tetraploid cells occurred in
the tumor transplanted to the B-strain at the first transplant generation,

RESEARCHES CARRIED OUT IN 1959

147

they showed no increase in this strain at the 5th and 6th transplant generations.
The chromosomes in 100 near-diploid and near-tetraploid cells werecounted exactly. Cells containing 40 chromosomes were at the highest
frequency (90%) in the diploid cell group, while those containing 80 chromosomes were most frequent (36.0%) in the tetraploid cell group. The rangeof chromosome number variation in the latter group is much wider thanthat in the former. A comparative idiogram analysis of diploid and tetraploid cells showed that the latter were derived from chromosome duplication of the original diploid assortment.

103.

Invasive ability of diploid and tetraploid tumor
cells of the Yoshida sarcoma(By Tosihide H. YOSIDA)

As shown in the preceding paper of this report, two kinds of Yoshidasarcoma cells, with near-diploid and near-tetraploid chromosome numbers,
were observed after transplantation to a B-strain rat.
The percentages of near-diploid and near-tetraploid cells in the ascites
tumor at the 3rd transplant generation were in the ascites form 26 and 69,
respectively. The ascites tumor cells were transferred into the peritoneal
cavities of W -strain rats by usual transplantation technique, and small
pieces of the lung of the same animal were implanted into the peritoneal
cavities of rats of the same strain. On the other hand, small pieces ofsolid tumor developed in the peritoneal cavity were transplanted into theperitoneal cavities of other animals of that strain.
In the case of transplantation of the ascites tumor, the number of tetraploid cells was reduced to 37.5 per cent, while that the diploid cells wasincreased to 57.0 per cent. One among four rats inoculated with lung tissues
developed an ascites tumor in the peritoneal cavity.
The frequency of
diploid and tetraploid cells in the ascites tumor, thus developed, was 8.2 and91.0 per cent, respectively. On the other hand, the ascites tumor developed by transplantation of tumor pulp taken from the solid tumor showedthat the frequencies of diploid and tetraploid cells were 93.0 and 7.0 per
cent, respectively.
The results of the above investigation indicate that the infiltration-abilityof the tetraploid cells of the Yoshida sarcoma was much stronger than thatof the diploid cells.

148

RESEARCHES CARRIED OUT IN 1959

104.

Double inoculation of hyperdiploid and hypotetraploid cellstrains of the Ehrlich ascites carcinoma andtheir invasion into organs(By Tosihide H. YOSIDA and Kurao ITO)

A hyperdiploid strain of the Ehrlich ascites carcinoma was obtained fromthe Children's Cancer Research Foundation, Boston, in December, 1958.
Since that time the tumor has been transferred from mouse to mouse forabout 30 transplant generations. In addition, a hypotetraploid strain of
the Ehrlich ascites carcinoma was obtained from the Zoological Institute,Tokyo University.
Table 1.

Frequency of hypotetraploid cells from double inoculation withhyperdiploid and hypotetraploid Ehrlich tumor cells.

TranspI. gener.

1

3

6

7

I

8

9

10

% of 4X tumor cells,

40.4

37.1

15.0

9.5

I

8.5

7.5

7.0

------------------'-------- - - - -200 cells were counted in each sample.

Simultaneous inoculation with the two different tumors, hyperdiploid andhypotetraploid Ehrlich ascites carcinoma, was undertaken in order to findout which cells have a stronger competitive ability. Almost the same
number of hyperdiploid and hypotetraploid tumor cells were inoculated
simultaneously into the peritoneal cavity of the same host. In order toestimate the activity of the tumor cells, the number of metaphasic cells
was calculated on the basis of 200 tumor cells per each transplant generation. It was evident that the number of tetraploid tumor cells gradually
decreased in the course of transplant generations. In the 10th transplant
generation the frequency of the tetraploid cells decreased to about 7 percent (Table 1).
In the 6th transplant generation following double inoculation, lung, spleen,
and liver were removed from the tumor-bearing mouse, and small piecesof those organs were inserted separately into the peritoneal cavities of
new hosts. This method made possible intra-peritoneal proliferation oftumor cells which had invaded these organs. In this way, three ascites
tumor cell populations in the 7th transfer generation were obtained fromthose inoculations. As shown in the table (Table 1), the frequency of
hypotetraploid cells is 9.5 per cent in the case of usual transfer technique,while it increased to 18.0, 19.0, and 16.0 per cent in the case oflung, spleen, and liver inoculation, respectively. In the next transplant
generation which was obtained by serial organ transfer, the number of

RESEARCHES CARRIED OUT IN 1959

149

hypotetraploid tumor cells increased to 32.0 and 26.2 per cent, respectively,in the case of lung and liver transplantations.
Based on the above investigations the conclusion may be drawn that thehyperdiploid tumor cells in the ascites tumor are much stronger in competition than the hypotetraploid, but the latter have a higher ability to invade
than the former.

105.

Effects of extract from Gentiana upon living plant tissue(By YO TAKENAKA, Yoshito OGAWA and Tom ODASHIRO)Japanese Gentian and Gentiana Japonica Pulverata, which are sold inJapanese drug stores, consist mostly of dried roots and rhizomes of Gentiana
scabra Bunge var. buergeri Maximowicz. Gentiana Japonica Pulverata is
powdered while Japanese Gentian is not.
Both materials were treated with meth:mol. After evaporation of thesolvent, a dark brown oil residue remained. The extract solutioIlS at
a concentration of Ig/2mL were used in the following experiments.
Water solutions at various concentrations (0.5-10J,;;) were prepared fromthe extract (50% concentration), and living roots of Allium scorodoprasum
var. viviparum and Vicia faba were placed in the solutions for from 2 to
30 hours. Some living root-tips taken from the solutions were squashedimmediately by the hydrochloric-acetic acid-orcein method, while others
were washed and soaked in tap-water for various lengths of time before
squashing_ III this report, results of observations of materials treated in
5% solutions of this extract for 2, 4, 6 and 24 hours are reported.
In general, the metabolic nucleus did not show structural changes asa result of short-time treatments, but with the increasing time of treatment the nucleoli appeared to be enlarged like vacuoles.
Short-time treatments at prophase sho',ved chromosome stickiness. In
long-tirae treztments the spiral structure of prophzse chromosomes became
conspicuous, and the chromosomes seemed to revert to the metabolic stage.
At late prophase, the chromosomes of short-time treatments appeared to be
somewhat sticky. In late prophase of long-time treatments double structures
became clearly recongnizable and some nuclei seemed to have reverted tothe metabolic stage.
At metaphase, "c-pair figures" were seen, but the chromosomes were
never shortened and thickened as after colchicine treatment. The longer
was the treatment time, the more c-pair figures appeared, and the c-pairchromosomes tended to become metabolic nuclei. Toward late metaphase,the number of nuclear plates increased gradually, and two sets of sister

150

RESEARCHES CARRIED OUT IN 1959

chromosomes showed loss of polarity, a comb-like appearance and a tendency toward the metabolic nucleus.
At anaphase, many chromosome bridges appeared and sometimes multipolar nuclei were observed. Even at telophase, chromosome bridges occasionally were observed. From short-time treatment, faint connecting fibres
were observed, but they were not present after long time treatment.
In many instances, the two daughter nuclei were close to each other withno cell membrane between, thus giving rise to binuclear cells. In otherinstances, there were some metabolic nuclei with doubled chromosome
number.
From the results of observations mentioned above, it may be concluded
as follows:(1) This extract decreases the visibility of the chromosome matrix.
(2) It weakens the function of the centromere.
(3) It disturbs the polarity of chromosomes.
(4) It decreases the visibility of the spindle and connecting fibres.
(5) It weakens the repulsion between sister chromosomes.
(6) It produces chromosome bridges at anaphase.
(7) It checks or retards the development of cell membranes.
(8) It produces binuclear or polyploid cells.
(9) It suppresses cell division.
In spite of the fact that Gentiana scabra var. buergeri is not poisonous
to the human body, the extract acts radiomimetically on root-tip cells ofplants. These phenomena suggest the action of arresting tumor-cell division.

106.

Effect of glucuronic acid on the growth of the animal embryo(By Yoshito OGAWA)

The effect of glucuronic acid on the growth of animals has been reported for rats (MATSUOKA, et al., 1957, 1858: NAKAYAMA, et al., 1957,
1958) and chick embryos (TUNOO, et al., 1957: USIZIMA, et al., 1958), but
the relationship between glucuronic acid and animal growth has not been
made clear. In this experiment, the growth promoting effect of glucuronic
acid on the early developmental stages of the embryos of Triturus jJyrrhogaster, BOlE was examined. Embryos of Triturus pyrrhogaster are auseful material for this kind of test since the developmental process istypical and is suitable for treatment with chemicals.
Immediately after fertilization, the embryos were raised in glucuronic
acid (containing Lacton 20%) or sodium glucuronate (1.0%, 0.1%,
0.01%, 0.001% and 0.0001%) solution at 20°C, and the body weight
and length of the developing embryos were measured.
Furthermore,RESEARCHES CARRIED OUT IN 1959151

the growth progress with respect to organ formation was investigatedhistologically. Glucose treatment at the same concentrations and nontreated groups were prepared for control.
It was found that sodium glucuronate and glucuronic acid showed asignificant promoting effect on the early development of embryos keeping
a normal balance in organ formation. Sodium glucuronate appeared to be
more effective than glucuronic acid in promoting growth and its mosteffective concentration was 100 mg.flOOO cc. This effect of sodium glucuronate was observed approximately on the 13th day after fertilization(Stage-32), but it did not last longer than two weeks. The difference
between sodium glucuronate-treated and non-treated plots seemed to correspond to five developmental stages, representing the time equivalent to
132 hours at 20°C, but no remarkable effect of this chemical was foundwhen the treatment was started on the 18th day afterfertilization (Stage-36).
It is noteworthy in this experiment that the stage at which sodium
glucuronate become effective falls always approximately on the 13th day
after fertilization (Stage-32), irrespective of the dosage and the developmental stage at which the treatment was started. This experimental result
is very similar to the effect of Kinetin (6-Furfuryl-aminopurine) on thegrowth of the embryo of Triturus pyrrhogaster (Y. Ogawa, 1959). A
remarkable promoting effect of sodium glucuronate and Kinetin on animal
cell division was already observed by the writer two years ago. The
relation between the above two chemicals with respect to growth promoting
effect during early developmental stages is now being examined.

107.

Development 0/ muscle protein in the regenerating limb
tissue 0/ Triturus pyrrhogaster, BOlE
(By Yoshito OGAWA)

DeHaan (1956), using actomyosin anti-serum, reported a treansient appearance of actomyosin in the regenerating limb tissue of Amblystoma
mexicanum 20 days after the operation. His finding is very interestingsince it indicates the existence of a precursor of actomyosin. On the other
hand, the formation of action precedes that of myosin in the early developmental stages of both the chick (Y. OGAWA, 1957, 1958) and Trituruspyrrhogaster (Y. OGAWA, 1958, 1959).
An experiment was carried out in order to find out whether the appearanceof actin in the developing skeletal muscle before the formation of myosin
is limited to early developmental stages or is a general phenomenon. The
formation of actin and myosin in regenerating limbs of Triturus pyrrhogaster
after amputation at the knee was investigated essentially by the same

152

RESEARCHES CARRIED OUT IN 1959

methods as those employed in the experiment with the early embryonal
stages.
G-actin and myosin were isolated from the skeletal muscle of Trituruspyrrhogaster by the method of STRAUB and SZENT-GYORGY (1951). Bothproteins at a concentration of 25 mg/m!. were injected intravenously into
rabbits at three-day intervals, until they reached a total of 100 mg. The
rabbits were bled by heart puncture on the fifth day after the last injection,and the sera obtained was raised to serological specificity by a resorption
test with saline extracts of liver, spleen and skin tissue. Titration withhomologous antigen was then carried out and the titer of sera was adjusted
to 1:512 before preparing the precipitin reaction with a saline extract of
granulated tissue of amputated limbs. A constant volume of sera and progressively decreasing amount of antigen were used for preventing a possible
failure due to excess of antigen.
It was found that actin and myosin first become detectable in the
granula of regenerating limbs 20 and 27 days after operation, respectively,
as showen in Table 1.
Table 1.

Determination of proteins in the muscle of the regenerating
limb of Tritttrus pyrrhogaster BOlE

* Antigen concentrations used ranged from 1: 10 to 1: 160 in tissue weight.
DeHaan's result may be due to a cross-reaction of his anti-serum with
developing actin, because the actomyosin anti-serum often reacts with actin,
as already pointed out by KESZTYUS et al. (1949).
Therefore, it is probably generally true that in the skeletal muscle actin
appears before the formation of myosin, both in early developmental stages
and in regenerating tissue.

RESEARCHES CARRIED OUT IN 1959

lOS.

153

Invariance of X-ray spectra at various depths in phantom*(By Sohei KONDO and Takesi KATO)

To make a perfect interpretation of the relative biological effectivenessof X rays in terms of linear energy transfer, it is necessary at the present
stage of radiation dosimetry to know precisely the X-ray spectra in the
exposed materials. For this purpose, measurement of the X-ray spectrumin acrylic acid resin plates of a cm, 1 cm and 5 cm thick has been carriedout with a scintillation counter. The plates were placed in front of a
sodium iodide crystal nil in diameter and gil in length. The X-ray
KXC-1S generator was operated at very low filament voltage, ca. 0.2 V, so
that the counter was able to count incident photons with no appreciablecoincidence losses. The spectra shown in Fig. 1 were obtained at 7.3 mfrom the tungsten target of the X-ray generator operated at 170 kVp withno external filter.
Acrylic acid
--Oem
1cm
- .. - 5cm

50

100

heY

Photon energy (keV)
Fig. 1. Change of measured spectra of 170 kVp
X-rays with various thicknesses of absorbers
placed in front of the scintillation counter.

We may conclude from Fig. 1 that the spectral distribution of X-rays
remains almost invariant at various depths in the phantom. This also willbe the case in living things except for points near bone or some otherhigh atomic number material. Therefore, the LET spectrum of the absorbed dose in X-rayed biological material is obtainable by calculation fromthe X-ray spectrum obtained outside the exposed material.
Fig. 1, however, shows only the relative change of the X-ray spectrum.
Corrections for scintillator escape effects and for statistical spread of pulse
hights from the photomultiplier must be made in the measured spectrum* This research has been partly supported by a grant from the Rockefeller
Foundation.

154

RESEARCHES CARRIED OUT IN 1959

to obtain the true X-ray spectrum. Since the contribution of the lattercorrection to a continuous spectrum is rather small, we have made only
corrections for escape effects using the Rawson-Cormack Matrix ll . The
peaks of the spectra shown in Fig. 1 are due to characteristic X rays oftungsten used as the target of the X-ray generator. Fig. 2 represents

10

100

Photon energy (keY)

Fig. 2. Comparison of corrected spectra of primary 170 kVp
radiation from Toshiba KXC-18 generator. A: filter 0.3mm
Cu+l.Omm AI. B: filter 1.0 Cu+1.0mm AI. C: spectrum
for filter B calculated from spectrum A.

some of the corrected spectra for external filters A (O.3mm Cu+1.0mm AI)
and B (1.0mm Cu+1.0mm AI). In Fig. 2, the spectra of characteristic
X rays of tungsten are shown separately, since the effects of pulse heightspread due to such monoenergetic photons are appreciable. The dottedcurve C is the curve calculated from the observed spectrum A by taking
account of the increase in absorption coefficients of filter B comparedwith filter A. From the good agreement of the calculated curve C withthe observed B, we may conclude that the spectra shown in Fig. 2 areclose to true spectral distributions of X-rays.
1)

E. G. Rawson, D. V. Cormack: A matrix to correct for scintillator escapeeffects. Nucleonics 16, No. 10, 92-97 (1958).

RESEARCHES CARRIED OUT IN 1959

109.

155

Silver-phosphate glass dosimeters and their application to Xray
dose measurements inside X-rayed mice and to separate
measurements of thermal neutron fluxes andgamma doses in the jRR-l Reaetor ll(By Sohei KONDO)

Rods (1 mm in diameter and 6 mm in length) and plates (10 x 10 x 0.2, 1.0
and 3.00 mm) of silver-phosphate glass were made by Dr. Ryosuke Yotota,
Toshiba Co., Kawasaki, and a fluorimeter (see Fig. 1) was built by Toshiba
Company following the author's plan given schematically in Fig. 2. As is

Fig:l.' Fluorimeter.

well-known, this special glass exposed to any kind of ionizing radiationsacquires the ability to fluoresce when excited by ultraviolet rays. The
amount of fluorescence is linearly proportional to the exposure dose. The
fluorescence fades very little at room temperature in over a month.
The above rod dosimeters are useful for conventional X-rays in therange from 5 r to 10 3 r with better than ±5% accuracy.
These rods were implanted in various organs of two mice for measure11
This research has been supported by the Education Ministry of Japan for Trial
Manufacture Research and the Rockefeller Foundation.

RESEARCHES CARRIED OUT IN 1959

156

ment of internal exposure dose distribution and exposed to X-rays of 170kVp
with no external filter. The results are shown in Table 1. The certainty
that the glass readings can be taken as they are, in spite of the sensitive
dependence of the glass reading per roentgen on X-ray energy, has beenassured through our finding!) that the X-ray energy spectra in an acrylicacid resin phantom at various depths have an almost invariant shape.
Thus, we may conclude from Table 1 that within error of ±5% the dose
distribution in X-rayed mice is constant.
Table 1.

Organ

Relative exposure dose inside various organs of mice exposed
to 50 r of X rays of 170 kVp with no external filter.
Back bone

TestisIntestine(under the)abdomen

Kidney1.04±0.03

1.02±0.03

1.02±0.03

1.01±0.03

1.01±0.05

Lung(on the)
hack

Brain

Spleen0.98±0.04

0.94±0.05-------

Relative
dose
Organ

Liver

------------------ - - ---------

Relative
dose
Note:

1.00±0.05

1.00±0.03

0.99±0.03

The average of the fluorescence readings of all the rods for two mice istaken as 1.00.

The measurements of thermal neutron fluxes and gamma contamination
doses were made with the use of glass plates of 10 x 10 x 3 mm with
Table 2.

Position
and
power
level
Thermal neutron
flux(n.cm- 2 ·sec- 1)

Measured thermal neutron fluxes and gamma ray
does-rates in Reactor JRR-1.
No.7 Hole (at 11.8cm fromgraphite surface); 40 kw!

No. 16 Hole;
5kw

by glass dosimeterby gold foils andion chamber*by glass
dosimeter

2.0(1±0.19)xlOB

2.5X10 B

5.2x10 10--- --------- !----------

Gamma contamination dose-rate(rjh)

460(1± 0 .12)

500

7 .OX 104

* These figures reported by the personnel of JAPAN Atomic Research Institutewere based on measurements carried out several months before the present
work was done.

RESEARCHES CARRIED OUT IN 1959•

different AgP0 3 concentrations (8%, 10% and 12%) in No.7 Hole (thermalcolumn) and No. 16 Hole of the JRR-1 Reactor, the latter being close to
the center of the reactor. The principle of the measurement was givenin the previous abstract 2 ). The observed results are given in Table 2.
The agreement between the results obtained by independent methods issatisfactory. The advantage of this method is that the dosimeters are sosmall that they can be placed among the substances to be irradiated and,in addition, that the absolute values of thermal neutron fluxes and of
gamma contamination doses can be obtained simultaneously simply bycalibrating the glass readings with a known gamma field.
The No.7 and No.16 Holes have been most often used for radiobiological studies and so the data on Table 2 will be useful for determination of thermal neutron fluxes and gamma contamination doses in such experiments.
1)
2)

S. Kondo and T. Kato: (in this annual report.)
S. Kondo: Ann. Report Nat!. Inst. Genetics, Japan 9, 134-136 (1959).

7312

,

157

PIIotomultipher

Retmnce glass holder

Fig. 2.

Schematic diagram of fluorimeter.

158

BOOKS AND PAPERS PUBLISHED IN 1959
BY STAFF MEMBERS
ENDO, T. 1959 Biochemical and genetical investigations of flower color in Swiss
Giant Pansy, Viola X Wittrokiana Gams. II. Chromatographic studies on anthocyanin components. Bot. Mag., 72:10-19
1959 Ditto. III. Dominance relations in F i hybrids, with special reference
to flower color and anthocyanin pigment constituents. Jap. Jour. Genet. 34:
116-124FUKUDA, M., T. SUGAHARA, R. HASHIMOTO and R. HASHIMOTO 1959 Effect ofradiation on blood pattern. (in Japanese) Rinsho-no-Nippon, 5 (8), 554-566
FUJII, T. 1959 Mutations in Einkorn wheat induced by X-rays. V. Recovery ofchlorophyll content in some mutants and double recessive segregants.
Jap.
Jour. Genet. 34:261-267
1959 Crossing experiments between basi-viridis II and other mutant
strains of T. monococcu?n. W.I.S. Nos. 9-10: 11
1959 Double recessive plants between chlorina and striata of T. monococcum. W,I.S. Nos. 9-10: 12-13
and S. MATSUMURA 1959 Radiosensitivity in plants.
III. Experimentswith several polyploid plants. Jap. Jour. Breed. 9: 245-252
FURUSATO, K. 1959 New hybrids of Citrus. The Heredity 13 (10): 50-53
1959
Some examples of seedless fruits produced artificially. Kajitsu
Nihon 4 (12): 25-26
1959 Seedless fruits of pomelo and grape produced by gibbe'ellin treatment Noko to Engei 14 (10): 51
HARADA, Y., T. AI and S. KONDO 1959 Tracing of 32P-labeled feed movement indigestive organs of trout. Station Report, Sizuoka-ken, Fisheries Exp. Vol.
1: 18
HARADA, Y. and S. KONDO
1959 Effects of agricultural chemicals on calcium
metabolism of pearl-oyster observed with use of '''Ca. Ibid. Vol. 1: 29
INAGAKI, E. and Y. TAZIMA 1959 An experiment to break up E/:l gene into separate components by X-rays (Abst. in Jap.) Jour. Seric. Sci. 28(3): 185
KATSUYA, K. 1959 Susceptibility of chlorophyll mutants of Einkorn wheat tostem and leaf rusts, Puccinia graminis and P. t1'iticina. W.I.S. Nos. 9-10:
14
KIHARA, H. 1%9 Japanese expedition to _the Hindukush. (The native place of
6x-wheat.) Proc. 1st Int. Wheat Genet. Symp. (l~j58): 243-248
1959 Considerations on the origin of cultivated rice. Seiken Zillo 10:
68-84
K. YAMASHITA and M. TA"IAKA 1959 Genomes of 6x speci2s of Aegilops.
W.I.S. 8: 3-5- , - - - - and -- - 1959 List of the collected materials of Aegilops
in Pakistan, Afghanistan and Iran by KUSE, 1955. W.I.S. 8: 11-19
KIMURA, M. 1959 Preservation of mutations in "Radiation Biology" (in Japanese)•,

BOOKS AND PAPERS WRITTEN BY STAFF MEMBERS•

159

Shokabo, Tokyo pp. 275-331
1959 Formulas for the calculation of effective population size (in Japanese) Kagaku 29: 610-611
KITAGAWA, O. 1959 The effects of X-ray irradiation on selection response. D.I.S.
33
KONDO, S. 1959 Some aspects of radiation biology in United States. Kagaku no
Rhoiki No.9: 66-69
MATSUMURA, S. 1959 Radiation genetics in wheat. Proc. 1st Int. Wheat Genet.
Symp. (1958): 112-133
1959 Radiation genetics in wheat. IV. Relation between genetic effects
and X-rays of different wave length of r-rays in a diploid wheat. Seiken Ziho
10: 11-21
1959 Genetic effects of thermal neutrons on wheat. W.I.S. Nos. 9-10:
8-91959 Genetic effects of X- and r-radiations on Einkorn wheat. W.I.S.
Nos. 9-10: 10-11
and T. FUJII 1959 Radiosensitivity in plants. II. Irradiation experiments with vegetatively propagated plants. Seiken Ziho 10: 22-31
and S. KONDO 1959 60CO r-irradiation and its use for plant
genetics. Conf. Uses of Large Radiat. Sources in Industry, Warsaw, 1959
. - - - - and K. KATSUYA 1959 Susceptibility of wheat nullisomics and theirgigas-plants to leaf rust. (in Japanese) Jap. Jour. Breed. 9: 54-55
and M. NEZU 1959 Effect of X- and r-irradiation on chromosome aberrations in Vicia faba. Seiken Ziho 10: 98-101
M. SHIMOTSUMA, T. FUJII and S. CHUDO 1959 Effects of X- andr-irradiation on grapes. (in Japanese) Jap. Jour. Breed. 9: 54-55
MORITA, T. and T. TOKUYAMA1959 Studies on genic action of ¥V-locus in
D. melanogaster. D.I.S. 33
NAWA, S., T. TAIRA., B. SAKAGUCHI and T. MORITA 1959 Influence of radiationon the control of cellular metabolism. Symp. Cell. Chern. 9: 39-46
NEZU, M. 1959 Relation of ploidy to radiation effect. W.I.S. Nos. 9-10: 12-13
OGAWA, Y. and K. FURUSATO 1959 Bitter taste response to the fruit of Citrullus
colocynthis Schrad. (in Japanese) Medicine and Biology 50 (3): 79-81
1959 Growth, differentiation and regeneration XII.
Development of
actin in the regenerating limb of Triturus phrrhogaster, Boie.
(in Japanese)
Ibid. 50 (5): 200-202
1959 Relation between the synthesis of actin and myosin in the early
development of Triturus pyrrhogaster embryo. (in Japanese) Kagaku 29 (6):311
- - 1959 Isolation of Citbittol, a bitter substance contained in the fruit of
Citrullus coloc1fnthis, Schrad. (in Japanese) Medicine and Biology 50 (2): 52-54
1959 Growth, differentiation and regeneration XIII.
Development of
myosin in skeletal muscle of Triturus pyrrhogaster embryo.
(in Japanese)
Ibid. 51 (3): 116-118
OGAWA, Y. 1959 Two chemicals promoting cell division in the Yoshida sarcoma

160

BOOKS AND PAPERS WRITTEN BY STAFF MEMBERS
cells.

Gann 49 (Supp!.): 97-98
1959 Taste-thresholds for Citbittol, a bitter substance extracted fromthe fruit of Citrullus colocynthis Schrad. (in Japanese) Medicine and Biology
51 (5): 187-190
1959 Growth, differentiation and regeneration XIV. Kinetin as a growthpromoting substance for the embryo of Triturus pyrrhogaster Boie.
(in Japanese) Ibid. 52 (2): 59-62
1959 Ditto XV. Effect of glucuronic acid on the growth of Trituruspyrrhogaster embryo. (in Japanese) Ibid. 52 (4): 178-181
1959 Glucuronic acid and animal growth. (in Japanese with Eng. resume)
Proc. Vth Glucuronic acid Research Conference. 1-5- and M. SHIMOTSUMA 1959 Bitter substance in the fruits of the hybrids(F[) between watermelon and colocynthe. (in Japanese) Medicine and Biology
52 (6): 255-257
and K. FURUSATO 1959 Bitter substances in the ripening fruit of Citrullus colocynthis Schrad. (in Japanese) Ibid. 53 (2): 69-71
OKA, H.I. 1958 Variations in temperature responses among cultivated rice varieties. Phyton, 12 (1): 1-11.
1958 Intervarietal variation and classification of cultivated rice. Indian
Jour. Genet. & PI. Breed., 18 (2): 79-89
and W. T. CHANG 1959 The impact of cultivation on populations ofwild rice, Oryza sativa f. spontanea. Phyton 13 (2): 105-117
OSHIMA, C. and O. KITAGAWA 1959 Effects of induced lethals on viability inheterozygous condition. D.I.S. 33
1959 Genetic variability of DDT and dieldrin resistance in wild typestrains of D. melanogaster. D.I.S. 33
SADO, T. 1959 Histological study of radiation sensitivity of male germ cells inthe silkworm (Abst. in Japanese) Jour. Seric. Sci. 28 (3): 186
SADO, T. 1959 On the restitution of silkworm spermatogenic cells after irradiation. (Abst. in Japanese) Jap. Jour. Genet. 34 (9): 306
SAKAGUCHI, B. and M. TSUJITA 1959 Role of cytoplasm in the manifestation ofgene. On the lethal yellow gene in silkworm. Recent Advances in Experimental Morphology ed. by K. TAKEWAKI, M. HARIZUKA and M. FUKAYA. pp.
272-282
SUGAHARA, T., K. TUTIKAWA and M. ONOUE 1959 Experimental animals for the
research of radiation injury. (in Japanese) Igaku no Ayumi, 29 (9): 566-571
SUGAHARA, T. and M. HORIKAWA 1959 Studies on the effect of radiation on living cells by tissue culture. (in Japanese) Symp. Cell. Chern. 9: 167-190
and M. OZAWA 1959 X-ray differaction study on muscle. Chemistry of
Muscular Contraction. ed. by. Y. KUMAGAI, Igakushoin, pp. 289-321-- and T. YASUDA 1959 Biological aspects of radiation hazards. (in Japanese) Progress in Radiology ed. by K. TSUKAMOTO and T. ADACHI, IshiyakuShupan pp. 103-120.,

BOOKS AND PAPERS WRITTEN BY STAFF MEMBERS

,
l

161

TABATA, T. 1959 A chromosome study in some human tumors.
Cytologia 24:
367-377TAIRA, T. and S. NAWA 1959 A note on the pigmentation in the eye. D.I.S. 33
TAKENAKA, Y. 1959 A picture book of morning glory. Hokuryu-kan, Tokyo.
TATEOKA, T. 1959 Chromosomes of five indian and ceylonese grasses species.
Curr. Sci. 28: 78-79
1959 Note of some grasses VII. Cytological evidence for the phylogenetic
difference between Lepturus and Monerma. Cytologia 23: 447-451
1958 Notes on some grasses VIII. On leaf structure of Arundinella andGarnotia. Bot. Gaz. 120: 101-109
1959 Lepturus and Monerma: a remarkable example of parallel development of gross morphology in grasses. Evolution 13: (3): 418-420
TAZIMA, Y. 1959 Radiation physiology of the silkworm with special reference
to the effect of radiation on germ-cells.
Recent Advances in Experimental
Morphology ed. by K. TAKEWAKI, M. HARIZUKA and M. FUKAYA (in Japanese)
Yokendo, Tokyo pp. 249-271
1959 Atomic age and Heredity (a review, in Japanese) Iden 13 (10): 17-21
1959 Genic changes presumably caused by alellic genes (a review, inJapanese) Iden 13 (6): 28-31-- 1959 Extreme sterility of silkworm males caused by irradiation (Abst.
in Japanese) Jour. Seric. Sci. 28 (3): 186---- 1959 On the stage of action of radiation-induced dominant lethals (Abst.
in Japanese) Jap. Jour. Genet. 34 (9): 313
TUTIKAWA, K. and K. TUTIKAWA 1959 Genetic study on variation of leukocyte
values after total-body X-irradiation in inbred strain and random-bred mice.
(in Japanese) Bulletin of the Experimental Animals, 8 (3): 84-94
TSUJITA, M. and B. SAKAGUCHI 1959 Functional relation among several genes
composing complex loci. On the E-complex loci in silkworm. R,"cent Advances
in Experimental Morphology ed. by K. TAKEWAKI, M. HARIZUKA and M. FuKAYA. : 283-291, S. NAWA, and B. SAKAGUCHI 1959 Studies on a silkworm poison emaneting from Tabacco plants. Proc. Jap. Acad. 35 (4): 180-185
TSUNEWAKI, K. 1959 A ring chromosome in an F i hybrid between wheat andAgropyron. Canad. Jour. Bot. 37: 1271-1276
and E. G. HEYNE 1959 Radiological study of wheat monosomics. I.
Differential sensitivity of monosomic X and the disomic to X-irradiation. Genetics 44: 933-946
and - - - 1959 Radiological study of wheat monosomics. II. Differential sensitivity of 16 monosomics and the disomic to a single dosage of
X-irradiation. Genetics 44: 947-954- - - - - and B. C. JENKINS 1959 Monosomic and conventional analyses of genes
in common wheat. Proc. IXth Int. Congr. Bot. 2: 404
YOSlDA, T. H. 1959 Chromosomal alteration and development of tumor, 1. A new
subline of the Yoshida sarcoma developed by chromosomal mutation. Zeit.
Krebsforsch. 63: 209-221

162

BOOKS AND PAPERS WRITTEN BY STAFF MEMBERS
1959 Chromosomal alteration and development of tumors, II. Chromo·
some conditions and invasiveness of the Yoshida sarcoma cells.
Gann 50.
233-238
1959 Tumors in lower vertebrates. Protein, Nucleic acid and Enzyme
4: 127-130,

ACKNOWLEDGMENTS
The editor wishes to acknowledge Dr. F.A. LILIENFELD and Prof. A.B. BUHDICK(Purdue University in U.S.A.) for reading the manuscripts. (C. OSHIMA)

,
.-.J

!

•

@lft3!~,+liJf5EfJT4~~ 9 1}

EiT,Fn 35 ~. 10 Jj 15 8I"fJ ffil]

E1Hil 35 'f 10 Jj 20 8~ff

~ ff1f';if:

jtq

1J1t

~:\'z:il:j'i;;~liJf5'eFJfi*J

..

I

m~1J
I@ :\'z:~AilIj1r

7(
~
ft: ~
)11 j'i;; ~ liJf o/E FJf i*J

¥£

;t:t:

m

**~~m\;[i-Wpjzll!T~
184~AilIjFJr :~ OO~3tmx~PMlI]t±*Jli:~~m\;[Lji1Jz1l!T184~ ff PJT

00 !L iA 1~ ~ liJf ~ fiJT

~w.JW,:==:!1I;m~EE

1,111

it . (::::'!11;) 77, 772

;~ .

""
t"

II

I

100

,

200

4(lG

60:)

800

lOpOm