NATIONAL INSTITUTE OF GENETICSJAPANANNUAL R~PORT
No. 9

1958

Published by

THE NATIONAL INSTITUTE OF GENETICS
Misima, Sizuoka-ken, Japan
1959

Annual Report

J

r

~,

of theNational Institute of Genetics
No.9,

1958

1
:~
i

Published by

The National Institute of Genetics, Japan
1959

CONTENTS
General Statement
Abstracts of diary for 1958.. . . . . . . . . . . . . . . . . . . . . .. .. . . . . . . . . . . . . . . . .
Staff" .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..
Council" .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ..
Research program for 1958 . . . . . . . . . . .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Joint researches supported by the grant from the Rockefeller Foundation
Foreign visitors in 1958 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Researches carried out in 1958

A. Genetics, biochemistry and cytology of insects
1. On the E-region composed of several functionally related genes(Tsujita and Sakaguchi) ..... . . . .. .. .. .. .. .. . . .. . . .. .. ....
2. Structure and function of pleiotropic genes of the silkworm
II. Further experiments to break up the EFll gene into separate components (Tazima and Inagaki)
3. On lethal NIl and Nl2 embryos of the silkworm (Tsujita)
4. Genetic and biochemical studies on the lethal yellow silkworm,
Bombyx mori. On the mechanism of hardening of the cuticule (Sakaguchi and Tsujita)
5. Genetic determination of tyrosinase and protyrosinase in the
blood of the silkworm, Bombyx mori II. On the nature of
the protyrosinase (Sakaguchi) .. .. . . . . . . .. .. .. .. . . . . . . . .
6. DDT-resistance of Drosophila pseudoobscura (Oshima)
7. DDT and dieldrin (DL) resistance of Drosophila melanogaster(Oshima) .. . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .. . .
8. Studies on the formation of Drosophila eye pigments (Tairaand Nawa)
9. Purine metabolism in Drosophila melanogaster II. (Morita)....
10. The chromosome number of an Indian wild silkworm, Theophilareligiosae, HelL (Tazima and Inagaki)
11. Studies on a silkworm poison emanating from tabacco plants(Tsujita, Nawa and Sakaguchi)
B.

Genetics, cytology and taxonomy of cereal crops and related plantgroups12. Studies on the nucleus-substitution and -restoration in the re-

1
1
2
4
5
7
9
10

10

11
13

15

17
18
20
21
22
24
25

CONTENTS

ii

13.
14.
15.
16.
17.
18.

19.
20.

21.
22.

23.
24.

ciprocal hybrids, Triticum vulgare x Aegilops caudata (Kihara)
Ecogenetical studies on Agropyron II. (Sakamoto).
.
Cytotaxonomic studies in Poaceae IV. (Tateoka).
Glutinous and non-glutinous isogenic lines in rice (Oka)
A systematic study of Oryza by statistical methods (a preliminary report) (Morishima and Oka).
Discrimination between Oryza sativa and O. glaberrima bymorphological characters (Morishima and Oka) .,.
A preliminary note on investigations of wild and cultivated ricestrains collected from the mountain region of Orissa state,
India (Oka, Chang and Narise)..
A study of the flowering time in wild rice (Sakai and Narise)
Observations on flowering and seed development in a wild
rice species, Oryza rufipogon (0. sativa spontanea) in Ceylon(Narise)..
Photoperiodic responses of Oryza species (Katayama and Oka)
Variation in photoperiodic sensitivity among and within populations of Oryza perennis and other wild Oryza species (Oka,
Hu and Chang).
Susceptibility of wild and foreign cultivated rice to blast fungus,
Piricularia oryzae. (Katsuya).
Susceptibility of chlorophyll mutants of Einkorn wheat to stem
and leaf rust, Puccinia graminis and Puccinia triticina (Katsuya)
.

29
32
33
34
35
37

38
40

42
44

47
48

49

C. Cytology and genetics of Nicotiana and some other plants
50
25. Cytogenetic studies of the genus Nicotiana XI. (Takenaka)
26. Cytological studies in the genus Euphorbia III. (Shimoyama) .. 51
27. Cytotaxonomic studies in Veronica and related genera I. (Yamazaki and Tateoka). 54
28. Development of seeds and embryos in three crosses between
diploids and artificial tetraploids (Furusato). 54
29. Frequency of 3x seedlings in several "triploid" varietites ofsugar beets (Matsumura, Mochizuki and Nezu)
. 56
_ 57
30. Heterostyly and pollen grain number in buckwheat (Doida)
31. Developmental studies in the genus Polygonum I. Microsporogenesis in Polygonum persicaria (Doida). 58
32. Developmental studies in the genus Polygonum II. Effects ofgibberelline on microsporogenesis of Polygonum fagopyrum(Doida),,, .. 59
33. Further studies on Cherry-red leaf in tobacco (Sakai and Iyarna)
. 60

"

iii

CONTENTS

34.

Sexuality in Rumex hastatus (Takenaka).. .. .. .. .. .. .. .. .. ..

D. Biometrical genetics and Problems of Breeding
35. Inheritance of competitive ability in rice (Oka and Sakai)
36. Effect of soil fertility on competitive ability in upland rice varieties (Sakai and Iyama), .,". . . . .. .. . . . . . . . . . . . .
37. Studies on the breeding behavior of wild rice (Oryza rujipogon
and Oryza perennis) (Sakai and Narise) ., " .. .. .. .. .. .. .. ..
38. Genetic variability in wild populations of Oryza perennis andO. rujipogon (Sakai and Narise), .. ..
39. Variation in anthesis of wild rice populations (Oryza rujipogon
and Oryza perennis) (Sakai and Narise),
40. Genetic studies on seed size in rice hybrids (Sakai and Pinto)
41. Inheritance of size and shape of grains in rice (Iyama, Morishima and Oka) . . . . . . . . . . . . . . . . .. .. . . . . . . . . . . .. . . . . . . . . . .
42. Polygenic mutation, induced by X-rays, in biometrical characters of Drosophila melanogaster (Yamada and Kitagawa) ... _43. The effects of X-ray irradiation on selection response (Kitagawa)
44. Breeding structure of poultry flock which maximizes the genetic
progress by selection (Yamada)
45. Performance test and selection efficiency involving different
environments (Yamada and Ito) . . .. .. .. .. . . .. .. .. .. .. .. .. . .
46. Evidence of significant interaction betvleen sire family and dateof hatching in egg production of chickens (Yamada)
47. A comparative analysis of body weight in two purebreeds andtheir reciprocal crossbreeds in the domestic fowl (Kawahara
and Ichikawa)
48. Influence of heterosis on viability of F 1 hybrids between two
breeds of the domestic fowl (Kawahara) . . .. .. .. . . . . .. . . ..

E. 1Vlathematical genetics
49. A maximum principle in the genetical theory of natural selection (Kimura) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
50. A gene frequency cline determined by selection and migration(Kimura) .. . . . . . . . . .. .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
51. Conflict between self-fertilization and outbreeding in plants (Kimura)
52. Inverse approach to the estimation of genetic load disclosed
by inbreeding (Kimura) .. . . .. . . . . . . .. . . . . . . . . . . . . . . . . .. . . . .
F. Biochemistry of pigments and other substances in plants
53. Biochemical genetics of flower color in Swiss giant pansies,

61
62
63
64
66
70
72
73
75
76
77
78
80

81
82

83
84
87
88

CONTENTS

iv

54.

55.
56.

Viola x Wittrockina, Gams (Endo), .. .. .. .. .. .. .. .. 90
Qualitative and quantitative analyses of bitter substances in
the ripening fruit of Citrullus colocynthis (Ogawa and Furusato), .. .. .. .. .. .. .. 92
Bitter substances in the fruits of F 1 hybrids between watermelon and colocynthe (Ogawa and Shimotsuma) .. .. .. .. .. .. 93
Anti-cancer activity of "citbittol A" (Ogawa), . . .. . . .. 94

G. Genetics and Biochemistry of microorganisms
57. Curly flagellar mutants in Salmonella (lino) .. .. .. .. .. .. .. .. ..
58. Cistron test of motility genes in Salmonella (lino) .. .. .. .. .. ..
59. Pteridine metabolism in Aspergillus oryzae (Nawa)

95
96
97

H. Radiation genetics in animals
60. Radiation mutagenesis in the silkworm IV. Independence ofradiation intensity as shown by induced sterility of spermatogenic cells (Tazima)
98
61. Radiation mutagenesis in the silkworm V. Further data onradiation-induced mutation rates in the silkworm (Tazima
and Onimaru).. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
62. Histological study of radiation sensitivity of spermatogenic cellsof the silkworm (a preliminary note) (Sado)
101
63. Studies on mutation rates after chronic irradiation in mice. A
preliminary report on sex ratio (Sugahara, Tutikawa andTanaka)102
64. Effect of radiation on living cells in tissue cultures (Horikawa
and Sugahara)
104
65. Unsuccessful attempt at in vitro cultivation of spermatogenic
cells of the silkworm (Sado)105
66. Influence of radiation on mitochondrial function in cellular
metabolism (Nawa and Sakaguchi)
106
67. Studies on genetic variation in response to radiation in mice
I. Effect of single doses of total-body X-irradiation on theleukocyte values of the peripheral blood (Tutikawa, Onoueand Tutikawa)108
68. Studies on genetic variation in response to radiation in mice
II. Strain differences in mice in the response of leukocyte
values to X-irradiation (Tutikawa and Tutikawa)111
I. Radiation genetics in plants
69. Radiosensitivity in plants (Fujii).. .. .. .. .. .. .. .. .. .. .. .. .. .... 114
70. Differences in radio-sensitivity and -mutability between cultivated and wild strains of Japanese morning glory (Kihara

CONTENTS

71.
72.
73.
74.
75.

v

and Sakamoto)
117
Relation of ploidy to radiation effect (Nezu) " .. .. .. .. .. .. .. .. 119
Genetic effects of S- and r-radiation on Einkorn wheat (Matsumura)
120
Genetic effects of thermal neutrons on wheat (Matsumura)
121
Genetical analysis of a few chlorophyll mutants in Einkorn
wheat induced by radiation (Fujii)
123
Radiation-induced mutation of the self-incompatibility gene in
Brassica (Rajan)
124

J. Genetics and cytology of tumors
76.
77.

An attempt to explain the origin of tumor cells with V- or
J-elements (Yosida) .. . . .. .. .. . . .. .. .. .. .. . . .. .. .. .. .. .. .... 125
Chromosome breaks in Yoshida sarcoma cells induced by Xirradiation (Tabata, Hirumi and Yoshida)127

K. Growth, differentiation and regeneration
78. Growth-promoting effect of kinetin on embryos of Trituruspyrrhogaster, Boie (Ogawa) .. .. .. .. .. .. .. .. .. .. . . .. .. ..
128
79. Mitosis-promoting activity of a pregnancy serum (Ogawa)
129
80. Synthesis of contactile proteins in the early development of
the embryo of Triturus pyrrhogaster (Ogawa) .. .. .. .. .. . . .. 131
81. Effect of Na-glucuronate on liver regeneration after partial
hepatectomy (Ogawa) .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .... 132
L. Technical Note
82. Simultaneous measurement of thermal neutron fluxes and rray dose in thermal column by silver-activated phosphateglass (Kondo)
134
83. The mechanism of serological reactions examined by using
steroids (Ogawa)
136
84. A simple method for the determination of chromosome number
in rice (Nezu)
138

Books and papers published in 1958 by staff members. . . . .. . . .. .. . . .. 139

GENERAL STATEMENT•,

This y~ar has been more fruitful than last year in the establishment ofresearch facilities, namely completion of two greenhouses. Rice Laboratory
and Isolation Greenhouse, Radiation Laboratory and the Second Mousery.
The Rice Laboratory was built for studies on the origin of cultivatedrice by a grant of the Rockefeller Foundation. This greenhouse has shortday installation comprising five small paddy fields (2.6 x 3.5 m). These fields
are equipped each with a sliding darkroom chamber, which is controlled
by astrodials. The Isolation Greenhouse is constructed for the isolation ofplants which are crossed or self-pollinated. It is air-conditioned and has
darkrooms for short-day treatments.
The Radiation Laboratory is not quite new. The old Isotope Laboratory
was renamed after the new addition of 2 rooms for X-ray equipments, oneroom for neutron (Ra-Be 500 mg) and another for r (134CS 6000 curie) sources.
Collecting tours were conducted by two members participating at the
project for the studies on the origin of cultivated rice. Dr. aka went toThailand and Prof. Hirayoshi of Gifu University went to Malaya and Java.
They could add many valuable wild and cultivated varieties of Oryza to
our collection. Crossing experiments among different varieties of wild andcultivated rice are now under way.
Using silkworms, mice, Drosophila, grasshoppers and tumor cells, animal
geneticists began studies on the genetic effects of radiation in animals.
Under the grants from the Ministry of Education, there are two projects
which are the joint work of many collaborators within and without theInstitute. One is the study on consanguineous marriage in Japan and theother is on the mechanism of manifestation of genes.
Several members of the Institute went to Canada and the United States
to attend the Xth International Congress of Genetics (Montreal), the 1st
International Wheat Genetics Symposium (Winnipeg) and International Congress of Radiation Research (Burlington).
The second summer seminar on genetics for the senior high school
teachers was again very successful this year. 67 teachers from 43 prefectures attended.

ABSTRACTS OF D. ARY FOR 1958
Jan.
Jan.

8.
16.

Committee meeting of Animal Genetics group.
Meeting of Tobacco Research workers.

ABSTRACTS OF DIARY FOR 1958

2

Jan. 27.
Feb. 14.
Feb. 20.
Feb. 21.
Feb. 22.
March 7.
March19.
March20.
March24.
April 5.
April 15.
May 7.
May 19.
May 21.
June 2.
June 3.
June 4.

Meeting of Rice research group.
64th meeting of Misima Geneticists' Club.
20th Biological Symposium.
65th meeting of Misima Geneticists' Club.
17th meeting of the Board of Councillors.
21st Biological Symposium.
Committee meeting of Animal Genetics group.
66th meeting of Misima Geneticists' Club.
Committee meeting of Rice Research group.
Scientific meeting of Rice Research group.
67th meeting of Misima Geneticists' Club.
Committee meeting of Rice Research group.
68th meeting of Misima Geneticists' Club.
Scientific meeting of Rice Research group.
69th meeting of Misima Geneticists' Club.
Board meeting of Japan Association of Poultry Genetics.
General meeting of Japan Association of Poultry Genetics.
Committee meeting of Rice Research group.
June 10. Scientific meeting of Rice Research group.
July 11. 22nd Biological Symposium
July 12. Meeting of the Editorial Board of "The Heredity" (lden)
July 21-24. 2nd series of Summer Seminar on Genetics.
7. Committee meeting of Animal Genetics group.
Oct.
Oct. 30. 70th meeting of Misima Geneticists' Club.
Nov. 10. Committee meeting of Rice Research group.
Nov. 11. Scientific meeting of Animal Genetics group.
Nov. 13. Committee meeting of Animal Genetics group.
Nov. 26. Scientific meeting of Rice Research group.
Dec. 2. Committee meeting of Rice Research group.
9. Scientific meeting of Rice Research group.
Dec.
Dec. 19. 71st meeting of Misima Geneticists' Club.
Dec. 23. Scientific meeting of Rice Research group.

STAFF
Director
Hitoshi KIHARA, D. Sc, M.J.A.
l"Wembers
1.

Department of JV[orphological Genetics:

STAFF

3

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., Assistant Head
Tsuguo T ATEOKA, D. Sc.

3.

Department of Physiological Genetics:
Chozo OSHIMA, D. Sc. Head of Department
Hitoshi KIHARA, D. Sc, M.l.A., Flora A. LILIENFELD, Ph. D.,
Toshifumi TAIRA, and Sadao SAKAMOTO

4.

Department of Biochemical Genetics:
Mitsuo TSUJITA, D. Ag., Head of Department
Yoshito OGAWA, M. D., Saburo NAWA, Bungo SAKAGUCHI, Seizo TSUDA,
D. Ag. (on leave of absence)
Tom ENDO, and Tetsuo IINO, Ph. D.

5.

Department of Applied Genetics:
Kan-Ichi SAKAI, D. Ag., Head of Department (on leave of absence)
Hiko-Ichi OKA, D. Ag., Assistant Head
Yukio YAMADA, AKIRA MIYAZAWA, and Takatada KAWAHARA
Shinya Jy AMA

6.

Department of Induced Mutation:
Seiji MATSUMURA, D. Ag., Head of Department
Tsutomu SUGAHARA, M. D., Assistant Head
Sohei KONDO, D. Sc., Kiyoshi TUTIKAWA and Taro FUJII

Part-time Stalf and Research Associates•

Kan OGUMA, D. Ag., Ex-Director, Emeritus Professor of Hokkaido Univ.
Yoshimaro TANAKA, D. Ag., D, Sc., M.l.A., Emeritus Prof. of Kyushu Univ.
Taku KOMAI, D. Sc., M.l.A., Emeritus Prof. of Kyoto Univ.
Yoshinari KUWADA, D. Sc., MJ.A., Emeritus Prof. of Kyoto Univ.
Sajiro MAKINO, D. Sc., Professor of Hokkaido Univ.
Kazuo FURUSATO

Research members under the Grant from the Rockefeller Foundation
Rice Research Group:
Yukio DOIDA, Tadao KATAYAMA, Keizo NEZU, Hiroko MORISHIMA, Takashi
NARISE, Mityuya NEZU and Shohachi SHIMOYAMA
Animal Genetics Group:

STAFF

4

Hiroyuki HIRUMI, Osamu KITAGAWA, Toshiteru MORITA, Toshihiko SADO
and Toshihide T ABATA

Department of Administration
Kunio SHIMIZU, Head of Department
Sumiyoshi Sumo, 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
24

Misima Branch of Hatano Tobacco Experiment Station
Masao TANAKA, Head

Assistants

5

Whole-Japan Association of Poultry Genetics
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
Seiji KAYA, President of Tokyo University, Vice-Chairman
Kan OGUMA, Ex-Director, Emeritus Professor of Hokkaido University
Seizo KATSUNUMA, President of Nagoya University
Yoshichi ASAM:I, Emeritus Professor of Tokyo University
Toshitaro MORINAGA, Director of National Institute of Agricultural Sciences
Hideo MATSUKUMA, President of Japan l\lonopoly 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

RESEARCH PROGRAM FOR 1958

5

Ichiro ISHIKAWA, Commissioner of Atomic Energy Commission
Daigoro MaRIwAKI, Professor of Tokyo Metropolitan University
Kunizo FUKUDA, Emeritus Professor of Tokyo University
Kenpo TSUKAMOTO, Director of National Institute of Radiological Sciences

RESEARCH PROGRAM FOR 1958
Department of Morphological Genetics
Genetics of silkworm (TAZIMA, TSUJITA and INAGAKI)
Studies on the food preference in silkworm (TAZIMA)
Radiation mutagenesis in silkworm (TAZIMA and ONIMARU)
Genetic effects of chronic irradiation upon silkworm (TAZIMA and O~HMARU)
Theoretical studies of population genetics (KIMURA)

Department of Cytogenetics
Cytology and genetics of tumors (YOSIDA)
Determination of sex and sex-chromosomes in animals (YasIDA)
Cytogenetics with tissue culture method (YasIDA)
Determination and differentiation of sex in 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)
Cytotaxonomic studies in Poaceae (TATEOKA)

Department of Physiological Genetics
Genetical analyses on insecticide-resistance of Drosophila (OSHIMA)
Physiological and genetical studies on the phenotypic expression of characters in Drosophila (OSHIMA and TAIRA)
Studies on the origin of wheat (KIHARA)
Studies on substitution of nucleus in wheat (KIHARA)
Studies on Agropyron (MATSUMURA and SAKAMOTO)
Right- and lefthandedness in plants (KIHARA and LILIENFFELD)Studies on stoneless pomegranates (KIHARA)
Physiological genetics of the reaction of Japanese morning glory to daylength (SAKAMOTO)

6

RESEARCH PROGRAM FOR 1958

Department of Biochemical Genetics
Biochemical genetics of insects and microorganisms (TSUJITA, NAWA andSAKAGUCHI)
Embryological and biochemical studies of silkworm (TSUJITA and SAKAGUCHI)
Radiation biochemistry of genetic materials (NAWA 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)
Genetics of virus (TSUJITA and TSUDA)
Studies on the fine structure of the cell (TSUJITA and TSUDA)

Department of Applied Genetics
Studies on breeding and genetics in poultry (YAMADA and KAWAHARA)
Polygenic mutation in quantitative characters of Drosophila (YAMADA)
Theoretical studies of plant breeding (SAKAI)Studies on competition between genotypes in plants (SAKAI and IYAMA)
Population-genetic studies of" Red-Rice" growing among upland rice (SAKAI
and IYAMA)
Genetic studies on "Cherry-red leaf" in tobacco plants (SAKAI and IYAMA)
Genetic studies on the migration activity in Drosophila (SAKAI and IYAMA)Polyploidy and sterility in fruit plants (FURUSATO and MIYAZAWA)
Analysis of genes responsible for hybrid sterility and hybrid break-down inrice (OKA)
Genetic studies of some physiological and agronomic characters in rice (OKA)
Radiation induced mutation in quantitative characters of rice (OKA)

Department of Induced Mutation
Studies on radiation-induced mutation rates in mice (SUGAHARA and TUTIKAWA)
Physicochemical mechanisms of radiation effects on living organisms (SUGAHARA)
Studies on radiation-protection in animals (SUGAHARA)
Genetics of mice (TSUTIKAWA)
Relation between the quality of radiations and mutations (MATSUMURA)
Radiation genetics of wheat and barley (MATSUMURA and FUJII)
Radiation genetics and its practical application (MATSUMURA)Radiosensitivity in plants (FuJII)

JOINT RESEARCHES SUPPORTED

Improvement of sugar beets by means of induced triploidy (MATSUMURA)
Measurement of X-, {3- and r-ray dosages (KONDO)JOINT RESEARCHES SUPPORTED BY THE GRANT
FROM THE ROCKEFELLER FOUNDATIONI.

Origin of Rice

Section 1. Collection and preservation of Oryza species (H. KIHARA)a. Number of species being preserved .... 18 species
b. Study-tour to Thailand (H. I. OKA)
c. Study-tour to Malaya and Indonesia (I. HIRAYOSHI)
Section 2. Morphology and physiology of Oryza species (S. MATSUMURA)
a. Taxonomic studies in the family Graminae (1'. l'ATEOKA)
b. Statistical taxonomy of Oryza species (H. MORISHIMA and H. I. OKA)
c. Anatomical studies in rice (1'. KATAYAMA)d. Susceptibility of wild and cultivated rice strains to blast fungus (K.
KATSUYA)e. Radiosensitivity of wild and cultivated Oryza species (1'. FUJII)
Section 3. Population-genetics in wild and cultivated rice (K. I. SAKAI)a. Estimation of genetic variability among and within populations of O.
perennis and O. sativa spontanea in Ceylon (K. I. SAKAI and T. NARISEJ
b. Estimation of the percentage of out-crossing by biometrical methods(K. I. SAKAI and T. NARISE)
c. Investigation of flowering time in wild rice (1'. NARISE)
Section 4. Genic studies in wild and cultivat=d rice (H. I. OKA)
a. Comparison between O. glaberrima and O. sativa (H. MORISHIMA and
H. I. OKA)
b. Crossing-experiments for the sterility of hybrids between wild andcultivated rice strains (H. I. OKA and H. MORISHIMA)c. Variation in seed dormancy and germinating capacity among wild andcultivated rice strains (H. MORISHIMA and H. I. OKA)
d. Variation in photoperiodic response among populations of wild rice (1'.
KATAYAMA, W. T. CHANG and H. I. OKA)
e. Investigation of rice strains collected from Jeypore Tract, Orissa,
India (H. I. OKA and W. T. CHANG)
Section 5. Cytogenetics of Oryza species (Y. TAKENAKA)
a. Karyo-type analysis of Oryza species (S. SHIMOYAMA)
b. Determination of chromosome numbers of Oryza species (M. NEZU)
c. Embryological studies in rice (Y. DOIDA)8JOINT RESEARCHES SUPPORTED

d. Cytological studies of haploid plants in rice (C. H. Hu)
e. Studies of trisomies in rice (T. TSUCHIYA)
f. Crossing-experiments of Oryza species (T. KATAYAMA and H. MORISHIMA)II.

Studies on genetic effects of radiation on animals
Section 1. Radiation mutagenesis in the silkvvorm (Y. TAZIMA)a.

Variation in mutation response to radiation of silkworm germ-cells.
(Y. TAZIMA and K. ONIMARU)
b. In vitro cultivation of silkworm spermatogenic cells (T. SADO)
Section 2. Mutation in mammals (T. SUGAHARA)
a. Studies on mutation rates after chronic irradiation in mice (T. SUGAHARA and K. TUTIKAWA)
b. Strain differences in mice in the response of leukocyte number to Xirradiation (K. TUTIKAWA)
Section 3. Cytology and cancer problems (T.H. YOSHIDA)
a. Behavior of grasshopper chromosomes in response to irradiation (inmotion picture) (S. MAKINO and Y. OHNUKI)
b. Chromosome breaks in Yoshida sarcoma cells induced by X-irradiation(T. TABATA and H. HIRUMI)
Section 4. Radiation biochemistry of genetic materials (M. TSUJITA)
a. Influence of radiation on mitochondrial function in cellular metabolism(S. NAWA and B. SAKAGUCHI)
Section 5. Population genetics (C. OSHIMA)
a. Polygenic mutation, induced by X-rays, in biometrical characters ofDrosophila melanogaster (Y. YAMADA and O. KITAGAWA)
b. Genetic consequences of irradiation and selection in a biometricalcharacter of Drosophila melanogaster (0. KITAGAWA and Y. YAMADA),FOREIGN VISITORS IN 1958

9

FOREIGN VISITORS IN 1958
Feb.

20. Prof. Richard KUHN (Max-Planck Institute for Medical Research).
Gave lecture on "Chemical Problems of Biological Resistance".
Yfarch 7. Prof. G. F. FRAENKEL (University of Illinois, U.S.A.). Gave lecture on "Insect Nutrition ".
May 1. Dr. R. F. CHANDLER, Jr. and Dr. J. G. HARRAR (The Rockefeller
Foundation).
June 28. Mr. L. H. CHEN and Mr. T. H. Su (Department of Agriculture
and Forestry, Taiwan).
July 11. Prof. K. NARAYAN (University of Mysore, India).
Sept. 20. Mr. J. G. SERIANO (Department of Botany, University of Philippines, Philippines) and others.
Oct. 9. Dr. N. PARTHASARATHY (F.A.O. Regional Office, Bangkok, Thailand)
Oct. 20. Dr. R. F. CHANDLER Jr. (The Rockefeller Foundation)
Oct. 23. Prof. A. G. MCCALLA (Dean, Faculty of Agriculture, University ofAlberta, Canada) and Dr. ]. HLYNHA (Grain Research Laboratory, Winnipeg, Canada)
Nov. 1. Mrs. Savitri SAHNI (Hon. Director, Birbal Sahni Institute of Palaeobotany, Lucknow, India)
Nov. 5. Messers J. HARAHOP, M. KOESRIN, P. PRAYURANONG and W. CROCKER(Colombo Plan students from Asian countries).
Dec.'S 3. Mr. B. G. SHAILCH (Agricultural Research Station, Dokri, West
Pakistan) and Mr. A. AHMED (Agricultural Research Station,
Jejgaon, East Pakistan)
Dec. 22. Mr. C. S. HUANG (Taiwan Agricultural Research Institute). Did
research work supported by the Colombo Plan.

•RESEARCHES CARRIED OUT IN 1958
A. GENETICS, BIOCHEMISTRY ANDCYTOLOGY OF INSECTS
1.

On the E-region Composed of Several FunctionallyRelated Genes(By Mitsuo TSUJITA and Bungo SAKAGUCHI)

Several cases of crossing-over between genes belonging to the E-region
have been found. Among these, the most clear-cut example is that between
ElI and EKP.
From the cross + x Ell +/+EK]J, individuals with the genotype Ell EKP/+ +can be obtained and these are phenotypically quite different from Ell +/+EKp
individuals owing to a position effect. As EllEKP/+ + larvae phenotypically resemble EI/+ or EI/EI larvae, they cannot be told apart on thebasis of appearance. The homozygotes from the EIIEK]J/++ larvae canbe distinguished. The phenotype of E also resembles that of EllEKP/ + +,but larvae with the genotype E/+ or E/E lack the supernumerary crescent patterns on the 4th segment. These facts suggest the possibility
that the phenotypes EI or E may be produced by a complex region consisting of two or more loci, such as ElI and EKP. To test this idea,
female pupae with the genotype EHEKP/EIIEKP were irradiated with Xrays and mated with normal males. In the next generation we obtained
various types of mutants, among them EI or E types. These type ofmutants can not be obtained by the irradiation of wild type individuals.
Consequently it seems that these mutants are induced by some change ina region composed of two or more loci, such as EHEKP.
ETC is a mutant obtained from a cross between females with the genotype EIIEKP which were treated with X-rays, and normal males. Larvae
with the genotype ETC/+ have supernumerary crescent patterns on segments 4 and 6, and larvae homozygous for grc have supernumerarycrescent patterns on the 4th, 6th, 7th and 8th segments and lack all abdominal legs (Fig. 1 A, B). It seems likely that the embryos with the
genotype EllEGa develop through the blastokinesis stage, at which timeembryos homozygous for EGa die but the others hatch out.
It is assumed that the ETC mutant was derived from a change at somelocus or loci other than the EH locus within the region EHEKlJ, and that
it is composed of EH and EGa. The fact that recombination between Ell

RESEARCHES CARRIED OUT IN 1958"

11

and EGa is difficult to achieve seems to be explained by some structuraldefect of the region. It is well known that all of the embryos homozygous for EGa die at the blastokinesis stage but that the EGa gene is maintained in the heterozygote EGal+. On the contrary, individuals homozygous for ETO are viable. Therefore,
if ETO were composed of Ell and EGa,some intimate functional interaction
might take place between Ell and ECa,just as with the position effect between
Ell and EKfJ.
Larvae of the heterozygote ElI1ECa
have supernumerary crescent patternson the 4th and 5th segments. Fromthe cross + X Ell 1EGa, one larva withthe genotype Ell EGa1+ and one normal
larva were obtained in addition to anumber of larvae with ElT and EGa
genotypes. Unfortunately, the embryosin the eggs laid by the EHECa type in- Fig. l. Larvae homozygous for ETo.
A: abdominal view
dividual died, so we could not study
B: dorsal view
them, but it seems that this recombinant individual is the same as that one known to have the genotype

E7'°I+.
In short, the E region of the silkworm is composed of several genes
separable by crossing over, and it is characteristic of this region that itis larger than similar regions on the chromosomes of other animals.
Furthermore, it may be considered that each of the genes composing theE region form a functional unit, at least from the viewpoint of size, andthat there are intimate interactions in functions of the genes of which theregion is composed.

2.

Structure and Function of Pleiotropic Genes of the Silkworm.
II. Further Experiments to Break up the Ef!l Gene into
Separate Components(By Yataro TAZIMA and Eiichi INAGAKI)

The attempt to break up a complex locus into several unit components
with the help of X-ray irradiation was continued. The EfJl character(additional crescent markings on the 4th segment and two pairs of extra
abdominal legs on the 4th and 5th segments) was used as material forthis experiment.

12

RESEARCHES CARRIED OUT IN 1958

Heterozygous E''il females were X-rayed in the pupal stage with 4000 r
and were crossed, after emergence, with untreated partners of a normal
strain, which has a pair of crescents on the 5th segment. Disappearanceof additional crescent markings and extra legs was carefully noted in theF 1 offspring. 185 exceptional individuals, 6.7% of the total individuals
observed, were discovered as pheno-variants with respect to the characters in question. They were classsified into
EH
47.0:'6
seven types, according to their phenotypes, as
shown in Fig. 1.
The results are summarized as follows.
EI-2oa) Disappearance of extra abdominal legson the 4th segment occurred most frequently,
EI-3
13.5
i.e., 71.3% (El-l, El-2, El-4, Fl-6, El-7).
b) The frequency of the disappearance of
EI-4
9.2
two pairs of extra abdominal legs on the 4th
and 5th segments was very low, i.e., 2.7%
(El-2, El-7).
EI-6
12.4
c) Erasure of additional crescent markingsonthe 4th segment occurred with the fre15.1
quency of 52.9% (El-3, El-4, El-5, El-6,
El-7).
EI-7
2.7d) The frequency of the erasure of crescentson the fifth segment as well as the additional
Fig. 1. Classification of
discovered pheno-varicrescent markings on the 4th segment was
ants into seven types.
30.2% (El-5, El-6, El-7).
Among the 185 individuals four were found to be new mutants induced
at the EEiI locus, and their progenies were maintained by successive backcrossing to the normal strain. The rest of the variants were due tophenocopy, modifiers, chromosome breaks, translocations , etc..
Eventhough a considerable variation was observed in regard to the penetrance
and expressivity of each mutant trait, the strains are characterized bydifferent kinds of particular modification of the ENl character. In two
strains most of El individuals lack extra legs on the 4th segment, whilein the other two strains the majority of El lack both extra legs andcrescent markings on the 4th segment.
Thus it seems likely that these mutants might have arisen from mutations which occurred at different unit sites of Efll locus. However, eachsite does not seem to act independently, because the modifications expressed were not confined to the character, presumably controlled by a specificsite. When a mutation occurred at one unit site, it may have affectedalso the expression of some other unit sites. If we assume the unit sitesof the Efll locus to be a, {3, r, 0, c, ... and a mutation to occur at the

RESEARCHES CARRIED OUT IN 1958

13

7 site, the expressed character (EZ) would be modified as follows.

a+S + 7/+0 +8+···

1 1

1

1 1

a+b" +c' +d" +e+··· =El' phenotype
3.

On LethaZ NZ l and NZ, Embryos of the Silkworm(By Mitsuo TSUJITA)

A certain mutant of the silkworm, NZ l which occurred spontaneously,
was discovered first by Y. TANAKA (1925). This type of mutant can also
be easily induced by X-ray treatment of a certain genotype. NZ l and NZ 2
are mutants obtained by such artificial induction. Larvae with the genotype NZr/+ and those with the genotype NZ/+ have almost the samecharacteristics, but as larvae with the genotype NZd + have some different
traits, they can be distinguished from the others. It has been determined that all of these mutants have a deletion of some part of the 14th
chromosome in the portion extending from the U region to the marker

odk.
Crossing-over experiments indicate that the order of the comparative
lengths of the chromosomal deficiency in the three types is Nl> NZ l > NZ 2 •It is generally known that a homozygote for a deficiency, even if it isvery small, dies at a c2rtain stage of its development. Moreover, thestage at which these mutants die varies according to the length of the
deficiency. As NZ, NIl and NZ 2 differ in the extent of their deficiencies it
was expected that they might also vary in the stage at which the lethal
effect was expressed. To test this point, hibernating eggs and artificially
hatched eggs of the three strains were fixed in hot water or with Carnoy's
fixative, and embryos homozygous for each of the NZ types were removed
by cutting open the egg shells. The stage of development was determined by examining whole embryos or microscopic sections.
The results are as follows.
Individuals homozygous for NZ die at a very early stage, during theformation of the blastoderm, as reported by ICHIMARU (1956). However,in the case of NZl/Nlr, young embryos of normal appearance are formed
which advance through diapause and die at the longest embryonic stage(Fig. 1).
Embryos homozygous for NZ 2 develop normally through diapause, andthey die during the period between the longest embryonic stage andblastokinesis. However, their development beyond the longest embryonicstage is abnormal, and as shown in Fig. 2, strikingly malformed embryo
were observed.
Although larvae with the genotype NZ/ + and those with the genotype

14

RESEARCHES CARRIED OUT IN 1958

Fig. 1. Lethal embryo homozygous
for Nl i .

Fig. 2. Lethal embryo homozygous
for Nl 2 •Ntd+ have similar characteristics, the cross-over value between odk and
Nt is 2.8 and that between odk and Nt l is 5.2. This is how we determine
the extent of the deficiency in each, and it is this difference which finds
expression in the different stages at which their homozygous embryos
die. The cross-over value between odl? and Nt 2 is 7.2 and as the lengthof the deficiency in Nt 2 is smaller than in Nt l , the development of theembryos homozygous for the former advances a little further than thatof the latter.
Larvae with the genotype Nlj + differ from those with the genotypes
Nt/+ or Ntl/+. It seems of interest to inquire why the characteristics
of Nt and Nt l are almost the same and why Nt and Nt 2 are different.
As mentioned before, Nt, Nt l and Nt 2 differ in the lengths of their deficiencies. Consequently, it is clear that the points of fusion of all three
are at different places on the 14th chromosome. It seems that a position
effect might be involved in the production of different phenotypes.
Thus it is shown, as suspected, that embryos homozygous for Nt l orNt, die at a far more advanced stage than embryos homozygous for Nt.
Embryos having the genotypes Nt/Nt l or NldNt 2 do not die at the stageof formation of the blastoderm but develop until about the longest embryonic stage or beyond. From this we learn that in heterozygotes between
two deficiencies the time of death is controlled by the partner with the shorter deficiency; for example, in the heterozygotes Nt/+, Nt,/+, and Nt 2 /+the normal chromosome overcomes the effect of the deficiency of its
partner.
A number of larvae with the Nt phenotype were obtained by irradia-

15

RESEARCHES CARRIED OUT IN 1958

bon of individuals with the genotype EHEKPIEHEKP. It might be inferred that all of these genes were involved in a deficiency in the abovementioned portion of the 14th chromosome. However, it seems that theextent of the deficiency is not always the same.

4. Genetic and Biochemical Studies on the Lethal Yellow Silkworm,
Bombyx mori. On the Mechanism of Hardening of the Cuticle(By Bungo SAKAGUCHI and Mitsuo TSUJITA)

Lethal yellow silkworm larvae ([emlilem l) which are produced by mothers of the genotype +Ilem l grow normally up to the youngest larval
instar, but they die immediately after the first moult because they cannot
feed. We have been interested in determining the exact cause of thismalfunction which is connected with imperfect differentiation of themandibular cuticle and incomplete hardening of the cuticular layer of thehypodermis. In order to determine the mechanism of cuticular hardening, various biochemical analyses were carried out.
(1) Quantitative analysis of scleroprotein and other proteins in yellow
lethal and normal larvae
Larvae of the two types were taken just after the first moult for biochemical analysis. The protein found in these larvae was divided into
three fractions, water soluble, alkali soluble and alkali insoluble (scleroprotein), using a modification of the method of HAKMAN (1953). The amountof proten was estimated as nitrogen. The results are shown in Table 1.
Table 1.

Solubility of proteins from + and lem l larvae.

+
Fractionsmg
Water soluble
Alkali soluble
Alkali insoluble
Total

89.5
16.0
108

116.3

mg

76.9
13.7
9.4
100.0

104.3
8.5
3.7
116.5

89.6
7.3
3.2
100.0

(Tests were made on 1 gram of dry material divided into three parts.)

Table 1 shows that the total amount of protein found in yellow lethals
is not different from that of normal larvae, but the water soluble proteinof the former was found to be greater than that of normal larvae. The
scleroprotein of the yellow lethal larvae, on the other hand, was shown
to be less than that of normal larvae.

16

RESEARCHES CARRIED OUT IN 1958

(2) Quantitative analysis of phenolic compounds
It is well known that the hardening of the insect cuticle depends upon
the conversion of a phenol substrate into a scleroprotein. The relationship between this hardening process and phenolic compounds in yellow
lethal larvae was therefore examined. A quantitative analysis of tyrosine
was made by the spectrophotometric method of CERIOTT (1957) and theamount of other phenolic compounds was determined by assay with
Folin's reagent. The experimental results are shown in Table 2.
Table 2.

Quantitative analysis of tyrosine and other phenolic compounds
in normal and lethal yellow larvae.
Tyrosine%

Water soluble
Alkali soluble
Alkali insoluble
Total and Total(Ty/N or Ph/N)

lem l

+

FractionsI

Ty/N i

85.6
21.2
9.0
20.0
5.4 I 20.7
100.0

Phenolic compounds

21.1

%

I

Ty/N

86.5 1'17~i
11 . 926. o·
1.6
10.7

+
%

lmn l

Ph/N

%

Ph/N

88.21~!88.6
7. 3.
6.5
100.0

25. 0 I
37.9
32.2

26.8
8. 2
30. 7
3.2 I 26.6

100.0

27.1

Ratio of tyrosine and phenolic compounds to nitrogen are shown in percent of
their substances contained in each fraction.

As is seen clearly in the table, the amounts both of tyrosine and of
phenolic compounds in the water soluble and the alkali soluble fractions
were greater in yellow lethal than in normal larvae. On the other hand,the amounts in the alkali insoluble fractions and the total amount of thesecompounds contained in yellow lethal larvae vvere less than in normal
larvae.
From these results, then, we conclude that the phenolic compounds, oneof the materials of scleroprotein are insufficient in the cuticular layer ofyellow lethal larvae, resulting in imperfect hardening of the cuticle.
From our previous work (1957, 1958) and the present results, it seems
evident that the lem l gene, which allows accumulation of a yellow pigment (Xanthopterin-B), primarily affects pteridine metabolism and is accompanied by a secondary effect upon the metabolism of phenolic compounds. This, in turn, results in a decreased formation of phenol substrates, and of scleroprotein. Softness and weakness of the cuticularlayer in the mandibular and other integumentary tissues are the net effects. Thus the yellow lethal larvae die because of their inability to
feed.

..

RESEARCHES CARRIED OUT I~ 1958

5.

17

Genetic Determination of Tyrosiilase and Protyrosinase in the Bloodof the Silkworm, Bombyx mori. IJ. On the Natureof the Protyrosinase(By Bungo SAKAGUCHI)

A mutant of the silkworm called ty, which shows low tyrosinase activity, causes the accumulation of more protyrosinase in mutant blood thanin that of normal strains. The nature of the protyrosinase contained inty larvae was found to be similar to the tyrosinase which is produced bynormal strains (SAKAGUCHI, 1958) by tests of the solubility in ammonium
sulfate and immunological reactions.
To determine the nature of the protyrosinase, further biochemical andgenetic studies were carried out.
(1)

Quantitative analysis of copper in the blood of normal and ty larvae

It is well known that copper acts as coenzyme of tyrosinase. In orderto determine any quantitative differences betw-een the copper contained inprotyrosinase and tyrosinase, the amount of that substance in both enzymes
in the blood of 5th instar larvae of the normal and the ty strains wasmeasured by the spectrophotometric method of Gubler (1953). The resultsof these experiments are presented in Table 1.
Table 1.

Copper contents of normal and ty larvae.
StrainSourceWhole blood
Whole blood (ashes)
Tyrosinase fraction
Non-tyrosinase fraction+ty

1.32±0.28, .23±0.28

1.43±0.35
1.17
0.50

1.51±0.41
1.:15

0.47
(i Icc blood)

As is shown in the table, the amount of copper contained i:1 the tyrosinase and non-tyrosinase fractions of whole blood shows ,'.1) significant
difference between the normal and the ty strains.
(2)

Quantitative analysis of sulfhydryl compounds

Tyrosinase activity is inhibited by reducing substances slIch as sulfhydryl compounds. Quantitative differences in the sulfhydryl compounds
contained in the tyrosinase and protyrosinase from the blead of normal

18

RESEARCHES CARRIED OUT IN 1958

and ty larvae were examined.
Quantitative analysis of the compounds was performed by measuring
the reducibility of allithiamine by sulfhydryl compounds. The results areshown in Table 2.
Table 2.

Quantitative analysis of sulfhydryl compoundof normal and ty strains.
StrainSourceTotal blood
Tyrosinase fraction
Non-tyrosinase fraction+ty

3.6±O.8
1.5±O.2
2.2±O.3

7.4±O.6
2.5±O.3
4.8±O.6(rice blood)

It is evident that in the sulfhydryl compounds contained in whole
blood, the tyrosinase and non-tyrosinase fractions of the ty strain weresignificantly greater than those of the normal strain.
It seems from the results obtained that the ty gene does not control
the amount of coenzyme (copper) in tyrosinase but controls the specificityof the apoenzyme. The mechanism of genetic control seems in addition,
to be connected with the quantity of the sulfhydryl compounds.

6.

DDT-resistance of Drosophila pseudoobscura(By Chozo OSHIMA)

This work was carried out at the laboratory of Professor DOBZHANSKY
in Columbia University. In natural populations of D. pseudoobscura; several kinds of inversions in the third chromosome have been observed; twoof these are Arrowhead (AR) and Chiricahua (CH). Two AR/AR homozygous populations, two CHICH homozygous populations and three AR/CH
heterozygous populations have been cultured for one year in population
cages. In the heterozygous populations, the frequency of both chromosomes has already reached an equilibrium; about 70% were AR and 30%
CH.
About 15 pairs of flies taken from each population were allowed to lay
eggs for three days in a bottle containing cream of wheat, molasses andyeast medium. Their 400 female offspring were divided into 10 groupsof forty flies each. These flies, 4-5 days old, were transferred to vials
containing a piece of test paper (5 X6 em") soaked with 0.5 or 1.0% DDT
solution, and were exposed to DDT for 10 or 15 hours. After exposure,RESEARCHES CARRIED OUT IN 195819

the flies were transferred to a fresh vial containing filter paper wet with
0.4 ml distilled water and the number of dead flies was scored 40 hours
later. The vials were kept in a room in which the humidity was about
50% and the temperature was 16°C. The results are shown in Table 1.
Table 1.

Accumulated mortalities of D. pseudoobscur-a
populations.

Population

Accumulated mortality 40 hours
after exposure to DDT

Mean (%)

180 ARjAR

35.8
51.3

43.5

47.0
39.5
42.0

44.9

30.0
16.5

27.3

175 ARjAR

181 ARjCH
176 ARjCH
173 ARjCH------------------182 CHjCH
177 CHjCH..

-

From the results of statistical analysis, no significant differences were
found in DDT-resistance between different chromosomal populations, but
the differences between popurations were significant. The CH homozygous
populations were more resistant to DDT than the AR homozygous ones.
About 15 pairs of flies were taken from the heterozygous populations
181 and 173 and 1000 F 1 flies were exposed to 1% DDT for 5 hours.
Forty hours after exposure, the surviving flies were cultured and theiroffspring were tested by the same method. Their resistance was foundto be increased (15% in mortality). After the test, 500 pairs of theiroffspring were transferred into a population cage and cultured for about
one month. The frequencies of both AR and CH chromosomal arrangements in populations were found by observation of salivary gland chromosomes of 150 larvae. The results are shown in Table 2.
Table 2.

The frequencies of AR and CH in populations
selected by DDT.

Population

Frequency of AR (%)

Frequency of CH (%)

181

80.3
87.0

19.3
13.0

173

- - - _ .. - - - - - ... -I

At the same time, the frequencies of both chromosomal arrangementsin the original 181 and 173 populations were examined by Prof. DOBZHANSKY20

RESEARCHES CARRIED OUT IN 1958

who found about 7096 AR. and about 309b CH in both populations. It was
assumed that the number of AR. was increased and the number of CH
was decreased by DDT selection.

7.

DDT and Dieldrin (DLJ Resistance 0/ Drosophila melanogaster(By Chozo OSHI~fA:)

The character of insecticide resistance has been generally assumed bygeneticists to be polygenic. Some factors in common to DDT- and DLresistance have been found in Drosophila by genetic analysis. Amongthem a dominant gene (or genes) was found to be located on the second
chromosome, but the mechanism of resistance to both insecticides wasproved by the following experimental results to have some different
components.
A highly resistant wild Hikone strain and a susceptible mutant strain,
having sea (scabrous) and ry (rosy) genes on the second and third chromosomes respectively, were crossed. The F 1 male flies were back-crossed
to sea; ry female flies and four kinds of phenotypes were obtained in theF 2 generation, as follows:+ (~e!L ry_), sca(~ca ry ), ry(sea_sea; ry(sea_ ".~)
+ +
sea +
+ rJ'
sea ryDDT- and DL-resistance in these flies was examined by using test papers(2% DDT, 0.4% DL), which were prepared by WHO in Geneva. Forty
female flies, 4-5 days old, were exposed in a small vial to DDT for 15hours and to DL for 1 hour; the accumulated mortality was observed ina fresh vial 25 hours after exposure. The experiments were replicatedthree times and the mean mortality was obtained. The results are shownin Table 1.
From the results, an important DL-resistant gene (or genes) was assumed to be located on the second chromosome and a powerful DDT-resistantlY),

Table 1.

DDT- and DL-resistance in F 2 flies of the hybrid
between Hikone and sea; ry strains.
Accumulated mortality (%)

Phenotype
DDT

sen
ry
sea;ry

DL

o

0.8

7.5

35.0

21.5

2.6
92.0

87.0

RESEARCHES CARRIED OUT IN 1958

21

gene (or genes) ,vas thought to be located on the third chromosome.
The latter finding is contradictory to the results previously reported. It
stands to reason that the homozygous ry flies are susceptible to DDT.
The ry gene inhibits the enzymatic activity of xanthine oxydase, which
has an intimate relationship to the respiratory system. Since the poisonous action of DDT affects this system, the ry flies appear to be more
susceptible to DDT. On this point the mechanism of the reaction to thetwo insecticides is different.

8.

Studies on the Formation of Drosophila Eye Pigments(By Toshifumi TAIRA and Saburo NAWA)

We have previously reported that the formation of red eye pigments
has an intimate relation to pteridine metabolism, because one of products
obtained by hydrolysis of the pigment was 2-amino-4-hydroxypteridine-6carboxylic acid. Several eye-color mutants were used for a comparison of
the relationship between the amount of eye pigments and of pteridines.
The results are summarized in Table 1.
Three kinds of eye pigments, red, yellow, and brown, were extracted
Table 1.
-- -

-----

--

--

-

Relative amount of eye pigments and pteridines.
-

---

-

--

-

-

-

-_

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

I- - . - - _ _~ _ _ _I -_---_._-

100

Body

Yellow Brown
YP

...

7

,

-----

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

Head

Strain

I

H.ed

--

Pteridine

Eye pigment

100

62

v

57

75

135
142

68
210

39
26

24
17

129
15

1); Hn j 3

3

100

122

S8

Hn T3

69

0

,

304

286

590

288

305

593

14

74
63

80

13

v;sc

33

4

73

Ty

6

V;Ty

6
0

0

82

bw
bw;se
v;bw

0

211

I

80

I
I

BFS

I

IXP

14

I

T

59

80

63

104

33
42

14
12

77

23

84

23

96

29

25
23
12

3

0

7
12

48

11

0

0
88

YP

14

5

3
0

T

__. _

Ore.-R

75

16
0

,

BFS

I
I

!

72
119
I

133

8

94

0

102

9

78

0

87

0

69
0

0

0

0

0

0

0

0

0

I

0
,

0
0
0
0
0
0
0
0
0
0
0
I
- - _.. _ - - - - - - - - Standard; Red and Brown: Oregon-R. Yellow: Se
YP: Yellow pteridine, BFS: Blue fluorescent substance, T: Total amount ofpteridine, IXP: Isoxanthopterin.
----- - -- --------~----RESEARCHES CARRIED OUT IN 1958

22

individually by a double extraction method, and their amounts were
measured photometrically. Several kinds of pteridines were eluted frompaper chromatograms, and the relative intensities of their fluorescencewere measured by fluorophotometer. The amount of eye pigment andpteridine were affected by the following conditions: (a) food, temperature,and crowding during the larval stage (b) age after emergence. The flies
were cultured under optimum conditions.
From the results summarized in Table 1, the following hypotheses canbe suggested. (1) Red and yellow eye pigments have an immediate relation to pteridines, because such strains as bw, v; bw, and bw; se, lacking
any red eye pigment, have no pteridines either in head or in body, butshow a trace of pteridine at the period of emergence. The bw gene inhibits the formation of red and yellow pigments. (2) It is a well-known
fact that the v gene blocks the formation of brown pigment derived fromtryptophane metabolism. The amount of red eye pigment of v is about
80 percent of that of the wild strain, although there is no abnormality inits formation. In double mutants containing the v gene, such as v; se,v;Hn r3 , and v;ry, the amount of red and yellow pigments are markedly
decreased. It seems that some interaction between the metabolisms ofpteridine and tryptophane might exist. In the case of v; ry, the amountof red pigment is about half of that of ry. It appears that strong effective interaction takes place in red pigment-formation under such restricted condition as ry. (3) The v;se and v;Hn r3 strains have a small
amount of brown pigment in spite of the v gene. This is assumed
to be due to an overproduction of brown pigment in both se and Hn r3
in comparison with the wild strain. (4) In the heads of se, Hn r3 ,v;se, and v;Hn r3 flies a large amount of BFS as well as of yellow pteridine (the main constituent of yellow pigment) is recognized. BFS consistsof two main components 2-amino-4-hydroxypteridine (AHP) and biopterin.
On the other hand, one component of BFS in Hn r3 and v;Hn"3 flies can
not be oxidized by xanthine oxidase and thus is not AHP, but it isthought to be an analogue of AHP. These mutants have also a yellowishgreen fluorescent substance which is catalyzed by xanthine oxidase into apurplish-blue one in the alkali range. The analyses of these substances
are now in progress.
9.

Purine Metabolism in Drosophila melanogaster II(By Toshiteru MORITA)

It is a well-known fact that the rosy2 (ry2) eye-color mutant of D.
melanogaster does not contain isoxanthopterin, which generally occurs in

RESEARCHES CARRIED OUT IN 1958

23

this fly. Neither does another rosy mutant, ryl, contain even a trace of
isoxanthopterin at any developmental stage. On the other hand, at pupal
and imaginal stages, large amounts of hypoxanthine and xanthine, precursors of uric acid, are produced, but no uric acid, which is the end
product of nitrogen-metabolism in the insect, is formed.
The purine content, detected by methods of chromatography and optical analysis at several developmental stages of Oregon-R and ryl strains,are shown in Table 1. It is very interesting to note that hypoxanthineis accumulated in the heads of adult ryl flies.
Table 1.

Purine content at various developmental stages
in D. melanogaster.

I

I

Oregon-R'ry

H~-poxanthi~~llliic ACid-Hypoxanthine Xanthine' U ric Acid
"gjmg

"gjmg

"gjmg

"gjmg

"gjmg
--------------

3rd instar larvae

0.0

0.06

0.1

0.10

0.0

Early pupae

0.11
0.06
0.05

0.70
0.80
1.10

0.85
1.55
1.85

0.28
0.48
0.52

0.0
0.0
0.0

0.0
0.65

2.63
0.37

0.0
0.19

0.0

1.38
0.20

0.0
0.20

0.0

Mid pupae
Late pupae-- - - - :---_._--

Adult,
male

head
body

0.33
0.03
-----

Adult,
femalehead
body-_.---~-----_._-

0.09
0.0

0.0

--~~~-I

0.0
0.30

I

0.0

_ . - - - _ .. -The enzyme, prepared from pupae, can catalyze the oxidation from 2amino-4-hydroxypteridine (AHP) to isoxanthopterin, as well as from hypoxanthine and xanthine to uric acid. This enzyme, therefore, has beencalled xanthine oxidase or pterin oxidase, but it is assumed to be a true
dehydrogenase because it requires electron acceptors such as DPN or
methylene blue for oxidation. As the homogenate can also catalyze theoxidation of DPNH to DPN, xanthine dehydrogenase seems to link a
DPNH-oxidase system by the medium of DPN in vivo.
The activity of xanthine dehydrogenase was not actually recognized inryl flies, but the activity of DPNH-oxidation could be distinguished.
A similar phenomenon was found in the double mutants v; ryI, en; ryl,
se; ryl, and stw 3 ; ryl. Both the deficiency of uric acid and the accumulation of hypoxanthine and xanthine in ry flies are assumed to be due tothe lack of xanthine dehydrogenase activity.
The activity of guanase, which catalyzes the conversion of guanine toRESEARCHES CARRIED OUT IN 1958

xanthine, was observed in pupal stages of both Oregon-R and ryl strains.
From these results, the process of uric acid formation in Drosophila IS
assumed to proceed according to the scheme illustrated in Fig. 1.
GUANINEl...cuanase

HYPOL~NTHINExrr. XANTHINE
I

,
I

ry

URIC ACID

Xanthine~ Dehydrogenase --=j;::--i-<

I

DPN

.

DPNH

DPN

~~
"----DPNH-Oxidase~
systemDPNH

JDPNH Oxidase

DPNH-Cytochrome C Reductase) Diaphorase

Fig. 1.

The pathway of uric acid formation and relative metabolic systems in

Drosophila.

10.

The Chromosome Number of an Indian Wild Silkworm,
Theophila religiosae Hel£.
(By Yataro TAZIMA and Eiichi INAGAKI)

The Indian wild silkworm is usually known as Theophila huttomWest. Adalbert SEITZ'S opinion (1933) is that the proper name of the insect is Theophila religiosae Hel£., according to the rules of nomenclature.
The larva of the Indian wild silkworm is quite similar to that of theJapanese wild silkworm, Theophila mandarina Moore except for the following characteristics: 1) the Indian wild silkworm has a larger bodythan the Japanese form and 2) in the former species each segment otherthan the 1st to 5th segments has a caudal horn-like nodule on the subdorsal line and a small nodule on the basal line, while the latter species
lacks the nodules completely. The larva of the Indian variety is trimoltic, as is that of the Japanese wild silkworm. The egg of the Indiansilkworm is about 1.3 times longer and 1.4 time wider than that of thedomesticated silkworm, Bombyx mori 1.. The cocoon of the Indian wild
form is yellow or pale green, and elliptic. The insect is found wild orrmulberry trees in south Himalaya and Assam.
We examined the chromosomes of the Indian wild silkworm. The

RESEARCHES CARRIED OUT IN 1958

25

Table 1. Observed data on chromosome number at metaphasein first spermatocytes.

~

No. of chromosomes
per metaphase plate I

No. of small chromosomes

l-liz-T3

L,__

4-5-6'-7_,_ '_Total metaphase
plates obsen-ed

31

3

9

9

1

1

o

36

30
29

o

o

220

1

5

1

1

1

o
o

o

4

13!

1

0

material was collected at Kalimpong, India, by the senior author m 1957.
Thanks to the kind assistance of Dr. D. C. Sarker, Deputy Director ofIndustries, West Bengal, the testes of the insect were fixed directly at
the habitat by Bouin's solution. Heidenhain's iron-hematoxylin method
was used for staining. n=31 chromosomes were found in the nuclear
plates of both first and second spermatocytes. The chromosomes differedin size, and three or four of them ,vere verysmall.
The domesticated silkworm which is no,,commercially reared in India and Japan has 28
chromosomes (n), while the chromosome number of the Japanese wild silkworm is 27 (n). It
therefore does not seem probable that the Indian.
Fig. 1. Ch,omosomes atwild silkworm might have been the ancestor of
metaphase in first spe~'­the domesticated silkworm in India, considering
matocyte.
the difference in chromosome number, althoughstudies on hybridization between the Indian wild silkworm and B. man
L. or T. mandarina have not been carried out yet.

, ....,......
.........
.,...-..

•

11.

Studies on a SiDhvorm Poison Emanating from Tobacco Plants(By Mitsuo TSUJITA, Saburo NAWA and Bungo SAKAGUCHI)

It has long been known that when silkworm larvae are fed on leaves
taken from mulberry trees near a field of tobacco plants, they are often
poisoned. It is clear from this fact that some poisonous substance evaporates from the tobacco plants and adheres to the mulberry leaves. There
are two different ideas resulting from studies on the nature of this poisonous substance. YABUTA (1931, '32) concluded that it was nicotine and
HASEGAWA (1931) insisted that it was trimethylamine. As it is necessary
to know the nature of the toxic substance if we want to protect silkworm
larvae from the damage caused by tobacco plants, the present studies

26

RESEARCHES CARRIED OUT IN 1958

were undertaken.
(1) The season of the emanation of the posionous substance fromtobacco plants.
Since 1956 special mulberry fields have been planted in which the trees
are placed in rows 3 meters apart from ridge to ridge (this distance istwice as wide as usual) and 0.76 meter between one stump and the next.
In the spring of every year young tobacco plants were planted mid way
between every two rows.
For the purpose of studying the season when the poisonous substanceemanates from tobacco plants and the silkworm larvae show symptomsof poisoning, experiments were carried out using leaves taken from thespecial mulberry field, and the following results were obtained.
i) The poisonous substance emanated from tobacco plants for a shortperiod during their maturity. According to records kept during the past
three years, the date of the beginning of emanation varies from year toyear. In 1956 it began about July 14, and in 1957 about July 9. As we
had a relatively dry rainy season in 1958 it was expected that poisonous
substance would be transferred earlier than usual. However it was delayed until July 29, and after few days the poison reached its peak.
ii) Judging from the symptoms of the silkworm larvae it appears thatthe poisonous substance is a kind of neurotoxin.
(2) Nature of the poisonous substanceTo determine the nature of the poisonous substance and to observe thetoxic effects of nicotine and trimethylamine on silkworm larvae several
experiments were carried out. The conclusion is that nicotine is far morepoisonous than trimethylamine to the silkworm larvae, and that thesymptoms of the two are different. Symptoms of poisoning displayed bysilkworm larvae which are mulberry leaves contaminated by the natural
emanations from tobacco plants markedly resemble those of larvae which
ate leaves painted with dilute nicotine sulfate solution or fumigated with
nicotine gas.
From these observations it seems clear that the poisonous substance isnicotine, not trimethylamine. Thus, our next step was to try to detect
nicotine and determine its amount in the poisoned mulberry leaves. Therefore, the extraction of nicotine from the following samples was carriedout. A) Normal leaves (control), B) Mulberry leaves fumigated with
nicotine gas (each mulberry tree was put in a wooden framework covered
with a polyethylene sheet and fumigated with nicotine gas), and C) Mulberry leaves contaminated under natural circumstances from adjacenttobacco plants.
The procedure for the extraction of nicotine from the leaves is as

RESEARCHES CARRIED OUT IN 1958

2T

follows:
Mulberry leaves 1 kg

t homogenized in O.lN HCI(4l)

filtrate

I concentrate to 500 ml in vacuo, and filtered after additiont of 10 N NaOH (20 m!)
filtratet extracted continuously with petroleum ether for 24 hours
pet. ether layer
textracted with 0.1 N HCI (10 ml)
aqueous layertsteam distilled, after addition of 10 N NaOH (1.5 ml)

distillate
Nicotine was collected on Amberite IRC-50 resin column (hydrogenform, 60-100 mesh) and then eluted with 0.1 N HCI.
Several fractions of the extract were used for chemical analysis.
From chemical analysis it was proved that the poisonous substance insample (C) is nothing more than nicotine.
(3) Nicotine content of the mulberry leaves
The nicotine content of several samples of mulberry leaves naturally
contaminated with nicotine to various degrees was calculated by themethod of Willits, Swain, Connelly and Brice (1950). The yield of eachof the samples was doubled because it was estimated that half of thenicotine content of the mulberry leaves was lost in the course of extraction. The results showed that nicotine content varies in different samples
from 1- 10 mg per kg mulberry leaves.
Next, when mulberry leaves painted with various diluted nicotine sulfate solutions (the ratio of the weight of the free nicotine to that of mulberry leaves was 1-10 parts per million) were given to silkworm larvae,
they showed various degrees of poisoning, ranging from very weak tovery strong. These symptoms, which vary according to the dose of
nicotine, are almost identical with those of larvae which fed on leaves
contaminated naturally to various degrees.
On the contrary, trimethylamine is so much less toxic to silkworm
larvae than nicotine and if want a violent response it is necessary togive far greater dose than with nicotine. We have not yet found trimethylamine in mulberry leaves which were naturally contaminated by
tobacco plants. If such leaves contain any trimethylamine, it must be a

28

RESEARCHES CARRIED OUT IN 1958

very small ari10unt. Consequently, its effect upon silkworm larvae may beconsidered to :Je negligible.
(4) Relative amounts of nicotine given off by several races of tobacco
plants.
It is quite clear that the chemical methods described above are indispensable to clarify the nature of the toxic substance. But the nicotine
content of the contaminated leaves is so small that it is necessary totake great care in its extraction as this procedure is troublesome. Therefore, chemical analysis alone does not offer a simple method of determining the degree of contamination of mulberry leaves, but our biologicalmethod, in \vhich the strength of response is observed in larvae fed onleaves contaminated with nicotine, is much simpler and more useful thanthe chemical method.
The relative amounts of nicotine given off by several races of tobacco,including Bright yellow, Enshu, Daruma, Matsukawa and White Burleywere determined by the above-mentioned biological method. The results
showed that the first four races emanate relatively large amounts of nicotine, but the last one an amount considerably smaller than the other.
(5) Experiments on the resistance of silkworms to nicotine
Various races of silkworm, including the Japanese races N 111, N 112,
N 115, N 122, N 124, N 501, the Chinese races S 108, S 110, S 115, S 124,
S 501, their hybrids, N 112xS 110, N 124xS 124, N 501xS 501, N 115x
S 108, and another race, Daizo, were examined for possible nicotine resistance. However, 2.3 far as the present experiment went, no conspicuous
differences were found among the races.
(6) ~icotine contaminatIOn of other plants.
Plants of the dandelion or fudanso (Beta vulgaris L. var. cida L), planted in flower pots were put near a field of tobacco plants during theperiod of emanation of nicotine, in July and August, and after few days
their leaves were given to silkworm larvae. It was found that theseplants were also contaminated with nicotine. This fact proves that otherplants including crops close to a field of tobacco would be contaminatedwith nicotine, although the amount is so small that it probably has noimportant effect upon human beings or domestic animals.
(7) Survey of the actual situation of the silkworm poisoning at a farm.
Recently we learned that silkworm larvae reared on a farm in Sizuokaken were poisoned by mulberry leaves grown near a field of tobacco
plants, and one of the authors, Tsujita, went to the farm to determine
the actual situation; about 4 kg of the damaged mulberry leaves werebrought back to our Institute for chemical analysis. The results of thisanalysis confirmed our experiments with mulberry leaves taken from the

RESEARCHES CARRIED OUT IN 1958

29

special field. Our observations and experiments are completely consistent
with the actual example of the poisoning of silkworm larvae which occurred at the nearby farm.
(8) Conclusion and consideration
It may be safely concluded from these experimental results that the
toxic substance, or at least its principal constituent, which emanates fromthe tobacco plants, mainly during their mature stage, is nicotine and nottrimethylamine.
It may be said that nicotine is given off principally from the leaves oftobacco plants. As the volatilization temperature of nicotine is 60-100 D C
and in the fields the temperature never rises so high even on summerdays, volatilization of nicotine from the surface of tobacco leaves is impossible. However, it is assumed that free nicotine molecules are contained in vapour evaporating from the secretions or from the disintegrating
cells of the hairs which cover the surface of the tobacco leaves, and itis this vapour which adheres to the leaves of other plants. Studies ofthe exact mechanism of the emanation of nicotine from tobacco plants
are now under way.

B.

GENETICS, CYTOLOGY AND TAXONOMY OF CEREALCROPS AND RELATED PLANT GROUPS

12.

Studies on the Nucleus-substitution and -restoration in the Reciprocal
Hybrids, Triticum vulgare x Aegilops caudata(By Hitoshi KIHARA)As already reported, T. vulgare (2n=42) and Ae. caudata (2n= 14) wereused in this experiment. Substitution and restoration of the nucleus werecarried out by successive back-crosses of T. vulgare as the male parent
to the reciprocal hybrids between the two species. Chromosome numbersof the first backcross generation (SB I and RBI) varied from 36 to 52. 49chromosome BI-plants were most frequent. Both 49-chromosome SB I- andRBI-plants had usually 21n+7r. To obtain Bz-plants only 49-chromosome
BI-plants were used.
In 1958 SB9 (9th back-cross generation of substitution line) and RB g (9thback-cross generation of restoration line) strains were obtained.
Chromosome conjugation in meiosis of both strains showed invariably
21 bivalents. Seed-fertility of both strains was normal; pollen-fertility was
zero in SB" resulting in complete male-sterility, and was perfect in RB g •Most of the RB 9 -plants showed all characteristics of T. vulgare. SB?-

30

RESEARCHES CAkRIED OUT IN 1958

plants, which are assumed to have the vulgare genomes in caudata plasma,
were in certain respects different from vulgare, namely as to male sterility
and right- and left-handedness of the spikelets.
Extensive studies of the karyotypes of both parents and plants of the
backcross generations (B-plants) were carried out.
a) T. vulgare is characterized by 2 sets of Sat-chromosomes, namely Sat1 and Sat-2. The remaining ones are V- or L-shaped.
b) Ae. caudata has in the haploid complement 2 Sat-chromosomes (C-Sat1 and C-Sat-2), 2J (J1 and J2) and 3 i-shaped chromosomes.
c) Karyomorphologically, the 4 Sat-chromosomes (Sat-I, Sat-2, C-Sat-l and
C-Sat-2) are quite distinct from each other.
d) The 3 i-shaped chromosomes can be identified by their size. However,identification is difficult if one or two are found in later generations.
e) The 3 i chromosomes of Ae. caudata can be easily distinguished fromvulgare chromosomes, when they are mixed with vulgare chromosomes.
However it is difficult to recognize the two J's, especially J1.
Using these morphological characteristics of vulgare Sats and caudatachromosomes, the writer could follow the progress of substitution andrestoration of the nucleus in the course of backcrosses. All Ba-plants haveattained the 6x-Ievel in this experiment. However this does not guarantee
complete genome substitution or restoration.
The effects of single caudata-chromosomes on the morphology of plants
could be in some cases clearly ascertained. For instance, C-Sat-2 is supposed to have a gene (or genes) for restoring male fertility in the substitution strains. This gene manifests itself when C-Sat-2 is substituted
for one vulgare-chromosome or added to the vulgare genomes. This gene(or genes) is completely linked with the gene for black awns.
In order to investigate whether or not fertility restoring genes can befound in other hexaploid wheats, T. spelta, T. compactum and T. macha
were crossed as the male parents with SBg-plants. Pollen-fertility of thesehybrids was as follows:a) SB s x T. spelta was male sterile, as was SBg •b) SBs x T. compactum was partially male fertile (seed-fertility by selfing
was 2.5%).
c) SBgx T. macha died before shooting. The same was observed when
T. vulgare and T. macha were crossed (KIHARA 1949).
Contrary to C-Sat-2, C-Sat-l has no effect on the morphology of B-plants
either by addition or by substitution. J1 is supposed to have a dominantgene for the waxy character of stems and leaves. Other caudata-chromosomes seem to have no effect when they are added or substituted singly
to vulgare genomes.

RESEARCHES CARRIED OUT IN 1958

31

First metaphase plates of PMC of several 42-chromosome B;l-plants having one C-Sat-l (or C-Sat-2) and 41 vulgare-chromosomes show almost always
2n. From these observations it was revealed that C-Sat-l conjugates eitherwith Sat-lor one of the non-Sat chromosomes of vulgare and C-Sat-2 conjugates either with Sat-2 or with one of non-Sat chromosomes of vulgare.
This relationship might be something similar to the homeologous relationship between the 3 wheat genomes, A, Band D.
As the pairing of chromosomes at MI of PMC of F l (T. vulgare x Ae.
caudata) varies from 0-6, complete pairing of C-Sat-l or C-Sat-2 with its
respective vulgare partner in substitution lines seems to indicate that thepairing is governed by genotypic control. The writer proposes the term
facultative conjugation for the pairing between two apparently semi-homologous chromosomes.
After careful observation of the stage following immediately the lastcontraction in Fl, maximum association of 28 chromosomes into 14 pairs
was found. Their number varied to a certain extent. However it is always
much larger than the number of bivalents at MI. This association is
dissolved mostly before early metaphase (Fig. 1 a-c).

a b cFig. 1.

Chromosome association in late prophase (a-b) and early metaphase (c) of T.

vu.lgare var. erythrosperm1tm x Ae. cmulata (F [).

Among 18 49-chromosome B1-plants, which were supposed to have arisen
from the unreduced female gametes of F l , 15 plants had the non-waxy
character of caudata. However 3 plants lacked the dominant gene for
non-waxy. As the genome-type of Bl should be AABBDDC, it was assumed
that crossing-over between the non-waxy caudata chromosome and one
vulgare chromosome took place when they were paired at prophase in the
Fl but later separated into two univalents.
The results were reported in detail at Xth International Congress of
Genetics in Montreal.

32

RESEARCHES CARRIED OUT IN 1858

13.

Ecogenetical Studies in Agropyron II.
(By Sadao SAKAMOTO)( 1)

F I hybrid between an early ecotype and the common type of A.

tsukushiense var. transiens Ohwi.
Artificial hybrids between a strain of the early ecotype described in aprevious report* and a strain of the common type were easily obtained.
The growth of the F I plants was very vigorous. Of the 19 observed
characters of the F I , 14(74%) were intermediate between the parents (Table
1). At meiosis of the F I hybrid, the frequency of tetravalent conjugation
Table 1. Characters of F I hybrids between an early eeotype and thecommon type of A. t8ukushiense var. tran8ien8 (1958).

Characters

Strains
Early eeotype(Cult No.) i(58107)----------- -- ----- -

-

-

-

-

--

Common type(58109)FI(5881)

I-- - - - - - - - -Upper side of rosette leaf

PubescentPubescent

Plant height (em)

67.22

1.67

3.41

27.85

0.53

85.85
40.16

1.83

39.70

3.55
1.23

11.75
14.96

0.40

11.21

0.39

0.44

18.11

14.29

0.45

18.65

8.86
0.85
0.20

0.16
0.10
0.01

6.68
0.80
0,16

0.40
0.41
0.21

3.43
0.19

0.27
0.02

3.61
0.17

1.15
0.13

0.07
0.02

1. 03
0.12

First internode from top (em),Flag leaf (em)

10.29

0.41

Spike (em)

11.12

0.03

No. of spikelets
No. of florets per spikelet

10.02
10.54

0.07
0.15

Length (em)Empty glume Width (em)

0.93
0.22

.
Length (em)Lemma WIth awn Width (em)

3.41
0.21

0.08
0.03
0.38
0.02

P I
Length (em)a ea Width (em)

1.28
0.15

Length (em)Seed Width (em)Weight (mg/100 grains)
Color of anther
A verage date of first heading
A verage date of first flowering ,---------

0.08
0.03
0.04
0.01
31.68

,

I

0.72
0.20
890.40red-brown

0.04
0.64
0.02
0.18
: 647.20 31.60
red-brown

26 April

7 May
9 May

I

I

28 AprilI
I

Non-pubescent
97.80

0.14
0.01
0.27
0.06

0.14
0.02
0.02
0.53
0.16
0
I
5.04
: 443.60
yellow----------

22 May24 May--------

amounted to 14% of the observed 163 PMCs. Pollen- and seed-fertilitiesof the F I were lower than in either parent but fairly good and the growthof the F 2 seedlings was very good.

* S. Sakamoto (1957) Ann. Rep. Nat. Inst. Genet. Japan 8: 34-36.

33

RESEARCHES CARRIED OUT IN 1958

( 2) Natural hybrids between A. tsukushiense var. transiens and A.
ciliare Franch.
A. tsukushiense var. transiens and A. ciliare are both common perennial
plants in Japan. The former is hexaploid (2n=421, growing in fields andalong road-sides, and the latter is tetraploid (2n=28), mainly occurring in
the hilly regions.
In the sympatrical habitats of both species in the suburbs of Misima,
11 clones of a natural hybrid between those two were found in the hills
and on river-dikes. The natural and artificial hybrids were morphologicallysimilar and somewhat resembled A. tsukushiense var transiens, but thelemmata were pubescent which is a specific characteristic of A. ciliare.
Both hybrids were vigorous but completely sterile.
Chromosome conjugation in the meiosis of the artificial hybrid showed
the configuration 9n+171 to 1511+51 (mode: 14n+71). Similar results had
been reported by MATSUMURA (1941),

14.

Cytotaxonomic Studies in Poaceae, VI.
(By Tuguo T ATEOKA)

1. In 1958, the chromosome numbers of the species listed in Table 1
were determined.
2. To examine the systematic relationship between Garnotia and Arundinella, their leaf structure was examined. In both genera unique features
were found which suggest that the two are related, but since their spikelet
structure is quite different, Garnotia may be treated as an independent
tribe, Garnotieae, tentatively placed near the Arundinelleae.
Table 1.
Species2nLeptaspis coohleata Thw.
Streptogyna crinita Beauv.
Arundinella villosa Am. ex Nees
A. leptochloa Hook. f.
Garnotia scoparia Thw.
Perotis patens Gand.
Spinifex littoreus Mear.

24
24
14
20
20
40
18

34

RESEARCHES CARRIED OUT IN 1958

15. Glutinous and Non-glutinous Isogenic Lines In Rice
(By Hiko-Ichi OKA)
Taichung No. 65 is a representative Horai variety of rice from Taiwan.
It is non-glutinous. In order to obtain a glutinous line of this variety,a strain was crossed with a Japanese glutinous variety, Kinoshita-mochi,
and back-crosses were repeated seven times using the same strain ofTaichung No. 65 as the recurrent parent. Then a heterozygote for theglutinous gene was selfed, and 22 non-glutinous and 22 glutinous homozygous plants were selected from the progeny.
The 44 descendant lines, together with the strain used as the recurrent
parent, were tested in a randomized-block experiment with three replications, at Taichung, Taiwan, in the first and second crops in 1958. There
were 135 plots in each crop, each consisting of 30 two-plant-hills. The
results of this experiment showed that the average yield of the glutinouslines (351.2 kgjha in the first crop. and 221.0 kgjha in the second crop) was
slightly lower than that of non-glutinous lines (357.3 kgjha in the first crop,and 222.3 kgjha in the second crop). The yield ratio of glutinous to nonglutinous was 98.3% in the first crop, and 99.4% in the second crop. But
variance analysis of the data showed that this slight difference was insignificant, as indicated below.
Table 1. Variance analysis of the yield of glutinousand non-glutinous lines of Taichung No. 65.
-

Variation due to- - - ---_..

Mean square

I

F

-

---

--

1%

5%

----

Replication
Class (Gl.: Non-g\.)Lines (within class)
Crop
Class x CropLine x Crop
Error
**

d.f.

----

Signi. level
-

2
1
43
1
1
43
178

14,824.0
923.0
1,114.7
1,188,428.0
391.0
1,210.4
I955.49I

15.52**
0.97
1.17
1243.78**
0.41
1.27

3.84
1.49

6.63
1.59

I.

Beyond 1 % level of significance.

As shown in the above table, it was also found from the variance analysis
that the differences among lines were insignificant. The degree of freedom
for error variance in this experiment was as large as 178, and there wasno reason for suspecting that this experiment was inferior to others in its
accuracy. It may then be assumed that all of the lines tested have

RESEARCHES CARRIED OUT IN 1958•

35

practicaliy the same genotype, except that the glutinous and non-glutinouslines differ in the glutinous gene and possibly some minor genes closely
linked with it.
It is known, as pointed out by MORINAGA (1943), that glutinous rice
varieties generally show lower yields than non-glutinous ones; the differenceis usually about 5%, and has been suspected to be attributable to thebiochemical nature of glutinous starch itself. The results of the present
experiment seem to suggest, however, that the glutinous gene may haveno such yield-Imvering effect. If this is true, it is conceivable that thegenerally low yield of glutinous varieties may be due to the effect ofcertain genes linked with the glutinous gene, which linkage might havebeen established in the course of origin of glutinous varieties.

16.

A Systematic Study of Oryza by Statistical .Methods*(a preliminary report)(By Hiroko MORISHIMA and Hiko-Ichi OKA)•This report deals with an attempt to find systematic relationships among
Oryza species by non-subjective techniques. The statistical method explored by SOKAL and MICHENER (1958) which deals with unweigted measurements of numerous characters was adopted for the study of 12 Oryza species.
First, correlation coefficients between species were computed from measurements of 20 characters, each of which had been normalized to have astandard deviation unit. Characters used are 1) grain length, 2) grain
width, 3) length x width of grain, 4) length/width ratio of grain, 5) apiculus
hair length, 6) empty lemma length, 7) ligule length, 8) awn length, 9)anther length, 10) pollen grain diameter, 11) weight of 100 grains, 12)
grain number per panicle, 13) panicle length, 14) number of first rachises,15) stoma size, 16) plant height at the seedling stage, 17) index of alkalitest, 18) germinating capacity (hulled seeds, 90 days after heading), 19)
number of days of germination, 20) seed fertility. Correlation coefficients
thus found between species ranged from 0.81 (0. sativa spontanea-O.
perennis) to -0.67 (0. sativa-O. eichingeri).
The matrix of correlation coefficients was then constructed from thedata, and the two species between which the correlation was highest in
both the row and column to which they belong were chosen to be the" nucleus" of the group to be formed. Then, the third species showing
the highest average correlation with members of the nucleus was added,
then the fourth species, and so on. The limit of a group was assumed* This work was supported by Grant RF 57080 from the Rockefeller Foundation.

36

RESEARCHES CARRIED OUT IN 1958

to be determined by a drop of more than 0.20 in correlation coefficient.
When the limit has been reached, a second matrix was constructed for
regrouping the resultant assemblage. Correlations between groups werecomputed by SPEARMAN'S sum of variables method. The same method ofgrouping as in the first correlation matrix was applied and matrices wereformed up to the fifth toO. ridley}obtain a single group of theO. ol'eviligulata
12 species. The resultant
relationships are shown inO. glaberrim:.La treelike diagram (Fig. 1.),
O. sativ&.
in which the magnitude ofO. sativa spontanca
correlation coefficient beO. perennis
tween any two joining stems
O. minuta
can be read on the abscissa.
We have some reservaO. officinali3
tions
about the results obO. eichi:,.geri
tained, however, because
O. latif01ia..
the number of characters" - - O. alta
used does not seem to be
O. australien3islarge enough, and theymight not be fully indicative-.6 -.4 -.2
0
.4
.6
.2
of the entire spectrum ofFig. 1. Diagram showing interspecific relationpotential variation amongships in Oryza, in terms of correlation coefthese 12 species. As Fig.
ficients.
1 shows,
however, thetreelike diagram thus obtained is not very far from the previously established classification of the genus Oryza, and may throw some new lighton phylogenetic relationships within the genus. For example O. rninuta
and O. ojjicinalis (r=0.79) are rather nearer to the group of cultivatedspecies and its relatives than to O. latifolia and O. alta, which form
another group together with O. australiensis and O. eichingeri. O. ridleyi
which has been thought to stand apart from the others, ,vas found to becorrelated with O. breviligulata which some workers have considered tobe the ancestral from of O. glaberrirna, though the correlation coefficient,
r=0.19, between these two species is too low to evaluate the relationshipbetween them.

••

RESEARCHES CARRIED OUT IN 1958

17.

37

Discrimination between Oryza sativa and O. glaberrima by
lvIorphological Characters*(By Hiroko MORISHIMA and Hiko-Ichi OK A)

A group or cultivated rice varieties grown in various regions of West
Africa has been regarded as belonging to a particular species, Oryz.aglaberrima Steud., which is characterized by glabrous glumes, short liguleswith a roundish tip, stiff panicles and other traits. However, it is inmany other characters similar to Oryza sativa 1., and the phylogenetic
relationship between these two species is not well known. To inquire
into the relationship between them, many rice varieties supposedly belonging to one or the other of these species were grown in Misima, anda number of morphological characters were investigated. 20 typical varieties of O. glaberrima and 30 varieties of O. sativa were chosen, and adiscriminant function for classifying the two species was constructed using
six morphological characters as given below. The linear function to maximize the ratio of variance between the two groups to that within thegroup was found to be:

X=xI-0.76x2+0.42x3-0.22xi+0.48 x5+2.70xu ,where Xl represents length x width of grain (mm 2 ), X2, length/width ratioof grain, Xs, empty lemma length (mm), Xi number of first rachises, X5,
panicle length (em) and XG ligule length (mm). The length of the apiculus
hair was nat included in this calculation since most strains supposedly belonging to O. glaberrima were hairless, and the species name "glaberrima"
itself implies glabrousness. It was found that the F value for the between-group variance of the composed character X was higher than thehighest value given by a single character (ligule length). The above
function would then be effective for discriminating between these two
Table 1.

Frequency distribution of the score given by a discriminantfunction for classifying O. sativa and O. glaberrima.
Score
10

45

50

Total numbpr
of varieties

Hairless varieties
Hairy varieties

3

2
12

1

33
51

Total

3

14 ! 184

Group

19 , 6. _ .. _-_.

2

10

__ _1

.....

16

13

_._-

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

38

RESEARCHES CARRIED OUT IN 1958

species. The distribution of the score X found in a number of varietiesof the two species is given in Table 1, in which the varieties are classified
into hairy and hairless.
The data in the table show that the two species can be cbssifieJ by
the score into two groups, though a few varieties appear to be intermediate.
Some varieties described in our collection-list as O. glaberrima wete foundto be near to O. sativa according to the score of this discriminant function.
A few varieties of O. sativa were hairless, but their scores were within
the range of that species.

18. A Preliminary Note on Investigation of Wild andCultiz'ated Rice Strains Collected from the 11.10untain
Region of Orissa State, India*(By Hiko-Ichi aKA, Wen-Tsai CHANG and Takashi NARIS E)
The mountain region in the western part of Orissa State, India, comprises about 10,000 square miles called the Jeypore Tract, which has neverbeen influenced by modern civilization. The greater part of this area isunder forest, but in valleys and some other places, rice is grown by thenative people in their "shifting" farms or in terraced paddies. It has
been found that this area contains an extremely rich variation in both
cultivated and \vild rice forms, and the Indian Council of Agricultural
Research has been since 1955 sending collecting parties to this area forsurvey and collection of rice strains. Through the kindness of Dr. S.
Govindswamy of the Central Rice Research Institute, Cuttack, India, who
is in charge of this project, one of the writers (Narise) could join thecollecting party and visit some parts of this area in November, 1957, when
he was staying in Cuttack with the senior writer. They were allowed touse the materials for their studies.
About 200 rice strains collected from the Jeypore Tract were tested by
the first two writers in Taiwan Provincial Agricultural College, Taichung,Taiwan. Seeds were sown on May 29, 1958, and five plants per strain
were raised in the experimental paddy. Investigation were made on an
individual plant basis regarding degree of grain shedding(S), grain weight(W), germinating capacity of grains 50 days after heading(G), resistanceto potassium chlorate(P), length-width ratio of grains(L), length of hairs
at apiculus(H), and many other characters.
Variations in these characters among the Jeypore strains were as large
or larger than those found among rice varieties widely collected from-------_.---* This work was supported by Grant RF 57080 from the Rockefeller Foundation.

RESEARCHES CARRIED OUT IN 1958

39

various Asian countries. In some cases, however, it was difficult to judge
from th:c appearance in the field whether the strains were cultivated orwild forrrs. In order to classify the wild and cultivated forms of rice as
clearly as possible, a discriminant function (Xl) was constructed based on
the variations in characters S, G and W among 21 Indian strains of Oryza
perennis and O. sativa spontanea, showing typical characteristics of thosespecies, and 21 cultivated varieties, including both the "Indica" and" Japonica" types. It was found that
Xl =S-1.857G-O.006,28W .

The distribution of the score given to the Jeypore strains by this formula
is shown in Table 1.
Table 1.

Distribution of the score Xl for discriminating wild andcultivated forms of rice.
Score
1.0

No. ofstrains

21

21
21

Population
1-1.8 -1.4 -1.0 1-0.6 -0.2

0.2

0.6

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

Cultivated vars.
Indian wild rice
J eypore strains
Group 1*
Group 2**

3

12

8

4

10

2

1
8

2
15

2
36

9
43

21
1

0~

oJ;:)

121

---------* Those considered by the collector to be wild.

**«

Those considered to be under human control.

The data in the table show that, although the 21 cultivated varieties
and the 21 wild forms can be clearly discriminated by the score, most of
the Jeypore strains are either intermediate between the cultivated and wildforms or nearer the wild form, and only a few of them are truly thecultivated type.
Secondly, in order to investigate the differentiation of the Indica (continental) and Japonica (Insular) types, a discriminant function (X2 ) which
may maximize the difference bet,veen the two types was constructed fromthe senior writer's old data (OKA 1957, Jap. Jour. Breed. 6), using characters P, Land H. It was found to be
X 2 =P-O.237L+O.567H .
Distribution of the score X 2 is given in Table 2.
The data in the table indicate that a greater part of the strains fromthe Jeypore tract, as well as of wild strains (0. perennis or O. sativa
spontanea), are intermediates between typical Indica and Japonica varieties,

40

RESEARCHES CARRIED OUT IN 1958
Table 2.

Distribution of the score for discriminating the Indica
and Japonica types.
Score

No. ofstrains
1-1.0 I-L~ -2.0 1-2.5 -3.0 i -3.5 -4.0 -4.5 I

Population

I,,--'--(---"

Cultivated vars.
"Indica""Japonica"
Jeypore strains
Group 1*
Group 2*
Indian wild rice**

2

12

20 ,

4
1

4

5
2
9

1
31

~I

4

22

7

40
35

,
,

31

3

5

10
40
2

13
40

3

7,

29
120
30

* Explanation in Table 1.
** Oryza perennis or O. sativa spontanea.

while a few of them are of the Japonica type.
in a more detailed study.

19.

The writers are engaged

A Study of the Flowering Time in Wild Rice
(By Kan-Ichi SAKAI and Takashi NARISE)

The flowering time of wild rice collected in India and Ceylon was studied.
The collection was made between Novembt'r of 1957 and February of 1958
at various spots in those countries. Seeds were germinated in petri dishE-s
in May of 1958 and young plants grown in pots were transplanted to a
field at Peradeniya, Ceylon, in June. The flowering time was measured
on an individual plant basis by the number of days from sowing of seeds
to the first day of flowering. Locations where the materials were collected
are as follows:
NotationofSpecies
Location
population
B
Oryza rujipogon
Chinsurah, West Bengal, India(0. sativa spontanea) P
Cuttack, Orissa, India
F-b
Cuttack, Orissa, India
T-(1-5) Samalkot, Andhra, India
III
Illuppaiyadichchenai, East coast of Ceylon
PP
Pottuvil Periphery, East coast of Ceylon
PT
Pottuvil Town, East coast of Ceylon
H-1
Oryza perennis
Cuttack, Orissa, India
Vey
Veyangoda, West inland of Ceylon
Yag
Yagoda, West inland of Ceylon* This work was supported by Grant RF 57080 from the Rockefeller Foundation.

..

•

..

Table 1.

.....

"

Flowering time of various populations of wild rice species.
-~

,

.~

-"! ~

,

aJ I 5..S I
e55' ' p':;.....

'"(j

J3
P
~ I
g,
I F-b

.

-"! :U,76

I '1'-5 ';""';1
III
"-

78

PP
PT

26

5:)

8

5

3

2

1
2

2

1

5
15 22

15
1

2
5
3

2

1
I

44 I 18·1
1j'~ I H-l3i9i"'(;;I Yag;- i,.
Ia i5.., Vey , 156
N ..,;,

2

11

'1'-4

a

10 " Mean
115 120 125 1:'30 135 140 145 150 155 160 165 170 175 180 185 190 195 200 205 210 215 220 •Z

21

;'
;1~

'"(jl

0

£. , '1'-1
1j-

zp..2 I I I I I I I I I I I I I I I I I I I I I I_1----=-:~ ___
58
76

~'

-

"6 -::: ~ 111 116 121 126 131 136 141 146 151 156 161 166 171 176 181 186 191 196 201 206 211 216 ~ 1::

1

I

-

1

6

8

1

2

,)

2
2

2

8

2

6

2
2
18
13

15 19
3 12
6 6
8 15

2

2

122.760138.55o 1134.21

1
12
13

10
14

11
13

12
5

4
7

9
16

9

5

7

7

5
1

5
1

2
2

1

1

2

4

2

1

0

157.92o 1159.87
0

1

2
8

o 1151.28

1i

152.34()o 1141.04
o ,140.91
i

4

12

10

9 70

1
119 64

24

21

17

9

14

20 17

6

-

-

15

7

3

1

20

5

4

1

1

69

-

1

0

182.6:1

2

0177.47

;>:;

rr1

if'

rr1

;p
;:d
(")

::r:

rr1

(f)

(")

;p

;:d

?::I

ti1

tj

0

c--:
>-3

2
r~

<J:)

CJI

00

-

*'"

I-'

RESEARCHES CARRIED OUT IN 1958

42

The flowering time of different populations of wild rice IS presented inTable 1.
Table 1 shows that among various populations of Oryza rufipogon (0.
sativa spontanea) native to India, there was a general tendency for plantsfrom higher latitudes to flower earlier than those from lower latitudes.
O. perennis flowered, in general, later than O. rufipogon. In this connection, it is noteworthy that most of the plants of the H-1 populationof O. perenttis collected in Orissa, India, did not reach anthesis within the
duration of this experiment, while all plants of O. rufipogon flowered before
the end. This indicates that the H-1 race may have become so sensitive
to a special photoperiodic cycle that they fail to flower under the conditions of Ceylon.
20.

Observations on Flowering and Seed Development in a Wild Rice
Species, Oryza rufipogon (0. sativa spontanea) in Ceylon. *(By Takashi NARISE)

Flowering and seed development in wild Oryza species have never been
observed in detail, so far as the writer knows. Wild rice plants belonging to O. rufipogon (0. sativa spontanea), collected from natural habitats
at Illuppaiyadichchenai and Pottuvil, Batticalo District, Ceylon, were grownin pots in the Botanical Garden, Peradeniya. At the same time several
cultivated varieties were also grown in pots, and their flowering and seeddevelopment were observed for comparison. Investigations were made on(1) how long the flowers remain open, (2) time of flower opening, and (3)daily increase in kernel weight after fertilization.
Results of observations on the duration of the open condition of flowers
are given in Table 1. As the data in Table 1 show, O. rufipogon and thecultivated variety indigenous to Ceylon showed not much difference in thisrespect, while the flowers of varieties introduced from foreign countries
remained open longer.
Secondly, variations in the time of flower opening In O. rufipogon anda cultivated variety, Mas M-24, observed on fine days and on rainy or
cloudy days, are given in Table 2. It is interesting to note that the wild
species showed a marked delay in flowering on rainy or cloudy days, while
the cultivated varieties did not depend so much on the weather.
Thirdly, the daily increase in fresh as well as dry weight of kernels in
O. rufipogon was observed. As the data in Table 3 show, it was foundthat the development of the kernels may be completed about two weeks* This work was supported by Grant RF 57080 from the Rockefeller Foundation.

..
Table 1.

Variation in the duration of flower opening in a wild rice species and five cultivated rice varieties.
Duration in minutes

Variety

OriginO. rufipogon*
Murugan Samba**
Mas M-24**
FAO No. 5817**
FAO No. 5818**
FAO No. 5820**
*
**

~

W

Ceylon3

Ceylon1

Indonesia

1

7

f/l

cO

~

,

41

9

11

10

3

1

2

3
9 : 16~114189110 j
14' 8
3Mean value

50

48.88 ± 6.13

8
1

1

50

43.88 ± 4.71

50

53. 32 j:: 9. 41

50

76.30 ±:: :;.49

::-:J

50

72 . 34 ±:: 6. 'l9

!JJ

50

68.24 + 5.24

In-'0'[ -45,1-0' [_1;'":;["";0'14' 15'

I'"

;p
::0
::0

...o ...-v
f/l

.QJ

O~

Mean value

Z'(l

2;-}

7

9

16 168 24

14

o -7 I 2

0 1 0

0

0

0

0

()

270

11!l04'Cloudyor rainy

o

o

o

66 68

2

10

6

9

2'

1

0

0

0

0

()

165

11 h 24'

Total

23

7

9

17 234 92

16

10

13

11

2

1

0

0

0

0

0

435

11h12'±20.30'

8

28

10

5

·16 47

35

16

19

18

5

6

2

7

o

2

255

1J1'24'

o o

()

o

3

4

42

43

15

6

131

12h 25'

8

10

5

386

llh48'±49.35'

47~O ~6 20 61 61 120 12
I

5

4

2

7 i 12

4

4

5

()

Average.i!
itemperature (OC) 26.227.4 28. 2 28. 928.828.828. 7 29.3,29.2,29.428.528.628.428.929.028.728.9
I

ti1
o
t:I

r:

f/l

Fair

28

n

~

i

'l:::..

tTl
;J>
::0

tr1

12h

h

tTl

!JJ

Variation in flowering time in a wild and a cultivated rice.

11

I

.)

Fair
" Cloudy
or rainyt;r TotaltI "'" g>;:"p.,

1(1);' .<lO', ·-45' -0' -1:;' -30' -4:;' -0'

:>

~

12

10

4 I 9
5 119

lOll

Weather

~

13

22
:;

~

Wild rice collected at IIluppaiyadichchenai.
Cultivated variety.

cO

N

12

6

Italy

...-

-oj<

4

Italy

:>,
h

~iOOlffiiroi~

Italy

Table 2.
.~~iOO

No. ofspikelets

t--J

S2
>-'

<n
vI

00

.".

v'

RESEARCHES CARRIED OUT I~ 1958

44
Table 3.

Daily increase in fresh weight, dry weight and water content ofdeveloping kernels in wild rice.

No. of days after
floweringoFresh weight (mg)

5.8

Water content (%)
Dry weight (mg)Ratio of dry to fresh
weight

2

3

4

7

8

6.0

4.9

6.0

7.7

14.1

11.8

45

38

33

38

4"J

36

46

40

35

3.2

3.4

3.313.7

4.4

5.8

6.7

8.4

7.7

.57

.64

.54

.59

.55

12

13

14

15

16

.55

~571~,_ .62

No. of days after
flowering

9

Will

Fresh weight (mg)

14.6

14.6

17.7

18.5

16.4

18.4

18.2

18.5

Water content (%)

31

27

30

30

36

24

24

24

Dry weight (mg)Ratio of dry to
fresh weight

.10.0

10.7

14.2

13.1

10.5

14.0

13.9

.68

.73

.70

.70

.64

.76

14.0

I

.761

.76

after anthesis. The water content of kernels seemed to decrease with theincrease in weight. The color of the glumes changed to brown about two
weeks after anthesis.

21.

Photoperiodic Responses of Oryza Species*(By Tadao KATAYAMA and Hiko-Ichi OKA)

The materials used in this experiment are strains of various Oryza
species collected from India and other foreign countries. This report isa preliminary note on the results of experiments on photoperiodic responsesof those species. For the strains from tropical countries, five automatically controlled short-day fields were constructed in this Institute with theaid of a grant from the Rockefeller Foundation. Three plots were set for
11, 12 and 13 hour-days. For the remaining two plots, special time-controlling clocks called Astrodial were used, with a "latitudinal control"
which brings about the natural change of day-length at a given latitude.
During 1958 the control was set for 35°N and the installations wereoperated so as to advance the calendar one month for one plot and twomonths for the other. Seeds were sown on May 19 and seedlings weretransplanted to the short-day field on June 23.

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

t

RESEARCHES CARRIED OUT IN 1958

45

In addition to this experiment, the same strains were grown in Taichung,Taiwan. They were sown on May 22 and June 21 and grown under conditions of natural day-length (see paper 22 by OKA et al. in this annual report), and photoperiodic responses, measured by the differences in the
Table 1.

Interspecific variations in photoperiodic sensitivity andcritical day -length.
Taichung

1---------------

No. of strains------_.-._----

Criticalday-length- - - --~--------

O. au straliensis
O. breviligulata
O. eichingeri
O. glaberrima
O. latifolia
O. minuta
O. officinalis
O. perennis
O. sativa
O. sativa v. spontanea
O. ridleyi

Sensitivity indegree

1
2
1
10
4
2
3
3
163*
4
1

10°
83°_90°13h 20'14h02'-14"09'90 0
75°_90°52°_70°78°_90 012°,40°_69°
83°_90°0°_90°

13h 23'1211 40'-13 b 23'
13 h 19'-14b 08'
13h 27'-13 b 30'
13h 45'-13h 58'
12h 59'-13h 20'
12h 45'-14h 30'
13 h 11'-13h 57'
13b 16'38 0 _70°
55°
MisimaNo. of strains

O. altaO. australiensis
O. eichingeri
O. glaberrima
O. latifolia
O. minuta
O. officinalis
O. perennis
O. sativa
O. sativa v. spontarwa
O. ridleyi* From OKA 1953.

1
1
1
9
1
1
2
5
6
7
1

Sensitivity indegreeCriticalday-length

6°
48°
58 0
56°_90°60°
3°84°_86°27°_67°
4°_68°
12°_76°12h 16'12h 15'b
12 OO'-13 h OO'
13h OO'I

non-heading I

12 lJ 58'-13 b OO'
12b 58'-13b OO'
I1h00'-13 h I9'
12h OO'-13h 30'
12 h OO'-13 h OO'

46

RESEARCHES CARRIED OUT IN 1958

length of the growing period, were recorded for the groups of plants sownin May and in June.
The sensitivity to day-length was measured by the method formerly
used by the junior writer (OKA 1953, 1958), i.e. by the ratio of shorteningof the growing period to a unit shortening of day-length at the time offlower initiation (assumed to be 30 days before heading;. The day-length
at which the ratio was largest was regarded to be the "critical daylength", and the sensitivity to that day-length was expressed in termsof the arc tangent of the largest ratio.
It is known that cultivated rice varieties (0. sativa) can be classified
into sensitive and insensitive strains. Almost all of wild rice material
tested, however, including many strains of O. perennis and O. sativa v.
spontanea, were found to be photoperiodically sensitive, except that an
insensitive strain was found in each of O. alta, O. officinalis and O.
minuta (Table 1). All tested varieties of O. glaberrima (cultivated) werealso sensitive, as shown in Table 1. This seems to suggest that photoperiodic sensitivity favors the wild rice plants with some selective
advantage.
It was found further that the critical day-length for wild rice strains
was 12 to 13.5 hours. The relation found between the critical day-lengthand the latitude of the place of collection is given in Table 2. The datain this table show the tendency for critical day-length to become shorterin low latitudinal countries. This may be due to the fact that heading
time is primarily determined by latitude in these species.
Table 2.

Latitude(N)

Geographical variation in critical day-length among photoperiodicallysensitive strains.
Critical day-length

12 15'

3
3
4

1

100-11 °13°_15°19°-22°24°_27°35°-37°~----_.-

hhZ-hoo'I

12 h 30'1 12 h 45' [ 13h OO'
I

2

I
I

II

1
6
7

I Tota.l of[ strams

3
5
12

I
I

:

13h 15' I 13 b 30'

1

8

2

2

-------------------- - - - - - - -RESEARCHES CARRIED OUT IN 1958

47

22. Variation in Photoperiodic Sensitivity among and within
Populations of Oryza perennis and Other Wild Oryza Species(By•

Hiko-Ichi OKA, Chao-Hwa Hu and Wen-Tsai CHANG)

A number of populations of wild rice belonging to Oryza perennis
Moench., O. sativa spontanea and other species, which had been collected
in India and Thailand by the senior writer, and in Taiwan by the juniorwriters, were grown in an experimental paddy in Taiwan Provincial Agricultural College, Taichung, Taiwan. The seeds were soaked in water for
germination on May 22 and June 21, 1958. Heading was observed ranging
from early September to early November, except for a few plants whichwere very late. The average temperatures of growing periods of different
populations are mostly between 26°C and 28°C, and the difference foundbetween May and June seedings in the number of days from seeding toheading can be attributed to the difference in the day-length to which
they have responded.
Photoperiodic sensitivities of those populations were shown, by themethod formerly adopted by the senior writer (OKA 1953, 1958), by theangle between the abscissa and the line showing the shortening of growing
period (in days) in response to shortening of the day-length 30 days before
heading (in minutes). Further, the day-length at the mid-point of the line
was considered to be the" critical day-length". Thus, the mean sensitivity and mean critical day-length of each population were determined.
Their standard deviations which show the extent of within-populationalvariation were computed from the standard deviations of the heading dateas follows: First, twice the standard deviation of the heading date was subtacted from, or added to, the mean heading date to estimate the early orlate marginal points of the distribution. Then, the early or late marginaldates for each of the two groups of plants belonging to a population,
seeded in May and in June, were compared by the above-mentioned method
to find the sensitivity and critical day-length of the earliest or latest plant.
Thus, a set of three measurements for earliest, average and latest plants
were found for each population.
The results of this calculation for each of the populations tested are
set out in Fig. 1, where the range of variation in photoperiodic sensitivity
or critical day-length is shown by a line connecting the values for theearliest and latest plants. It is recognized in Fig. 1 that some populations
are quite heterogeneous for photoperiodic sensitivity, while a few are uniformly highly sensitive. It seems that populations of O. sativa spontanea'" This work was supported by Grant RF 57080 from the Rockefeller Foundation.

48

RESEARCHES CARRIED OUT IN 1958
O. sativa spontanea
WOlOS
WOIOS
\\'0107
WOli52
WD153

O. perennis or spontanea
WOl:l~\\'() 1;-:.~ tW01;>,1\YOl;~5\\,Ol;3fi
WO[;10O. peren:lis

wol09
Wall:]
W0114
WallO
W01l7
WOll8
\V0145Wild rice of Formosa
AB

C
O. ridle~ri
WOODIo. officinalis (\\'0002): Insensitive
30°

40°

5Q.~

(1)0

70'"

80'"

90>

12"30'

13'

13'30'

14 11

Fig. 1. Variations in photoperiodic sensitivity and critical day-length
among polulations of O. perennis and other wild Oryza species.

generally show a wide range of variation in photoperiodic sensitivity, while
O. perennis is relatively uniformly sensitive and its variation in heading
date is chiefly due to variation in the critical day-length. It was foundfurther that O. ridleyi was as sensitive to day-length as O. perennis, andO. ojficinalis (a strain from Bangkok, Thailand) was insensitive.

23.

Susceptibility of Wild and Foreign Cultivated Rice to Blast
Fungus, Piricularia oryzae*(By Keiz6 KATSUYA)

In order to examine the susceptibility of the genus Oryza to blast, 14
strains of 9 wild species, 10 foreign strains of cultivated rice belonging toO. sativa and 3 varieties of Japanese cultivated O. sativa were tested
in a greenhouse with 2 strains of Piricularia oryzae, namely P-2 and 54-04,
obtained from the National Institute of Agricultural Sciences. As topathogenicity, the P-2 strain is comparatively virulent while the 54-04 strainis less virulent. The plants were kept in pots to which ammonium sulphate, calcium superphosphate and potassium sulphate were added. They

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

•

RESEARCHES CARRIED OUT IN 195849

were inoculated by spraying an aqueous spore suspension of the fungus
on the third or the fourth leaf, and by injecting it into the still folded
fourth to sixth leaf with a hypodermic syringe. The spores were cultured
on boiled barley. The degree of susceptibility was determined by thetypes of lesions formed on the leaves. Blast readings were made on
infected leaves 5 to 11 days after inoculation. Sheath inoculation (SAKAMOTO 1951, TAKAHASHI 1957) was used for comparison. These tests wererepeated 2 to 4 times.
The experimental results in general showed that the injection method
effected a more severe pathogenicity than spraying. Among the wild
species, O. australiensis (W 0008) was very susceptible to the two strains of
blast fungus. O. officinalis? (W 0012), O. eichingeri? (W 0015), O. latifolia(W 0019) and O. breviligulata? (W 0028) exhibited resistance to the P-2
strain when sprayed and resistance or moderate susceptibility when injection was used; they were resistant to the strain 54-04 when inoculated byinjection. O. subulata (W 0510) was moderately susceptible to the P-2
strain and resistant to the 54-04 strain.
O. perennis, O. sativa var. fatua and other foreign cultivated rice
varieties of O. sativa exhibited considerable differences according to thestrain employed.

24.

Susceptibility of Chlorophyll Mutants of Einkorn Wheat to Stemand Leaf Rust, Puccinia graminis and Puccinia triticina(By Keiz6 KATSUYA)

In order to examine the susceptibility of chlorophyll mutants of Triticum
monococcum flavescens to stem and leaf rusts, the mutants chlorina, basiviridis II, virido-albina and albina were tested in the phytotron (20°C in
daytime, 15°C at night) at the first leaf stage with Puccinia graminis f.
sp. tritici 17 and P. triticina 21 B. Both filter paper and brushing inoculation methods were used. Rust readings were made 7 to 16 days after
inoculation. The degree of susceptibility was determined by the types oflesions formed on the leaves. These tests were repeated 2 or 3 times.
Susceptibility of chlorophyll mutants to stem and leaf rusts is shown in
Table 1. The mutant strain" chlorina" whose chlorophyll content was
about 50% of that of the normal plants was not much different from thenormal plants in susceptibility to both rusts. However it showed a tendency to earlier formation of uredosori. The mutant strain "albina" differed from the normal plants in susceptibility to both rusts exhibiting
resistance. No uredosori were found, since albina dies from 7 to 12 days
after inoculation. Both kinds of rusts formed uredosori on the white parts,

50

RESEARCHES CARRIED OUT IN 1958
Table 1.
-

Susceptibility of seedlings of chlorophyll mutants to stem andleaf rust of wheat.

--~~=~-

I--~--

Type of Infection
Plants inoculated
I

NormalChlorina
green parts
I
Basi-viridis II { I' h
Ig t green parts
green parts
I
Virido-albina { h'w Ite parts
AlbinaP. graminis

P. triticina

f. sp. tritici 17

218

R-MR
R-MR

S
S
S
S
S
S

MR-S
MR-S

R

R

R: Resistant, MR: Moderately Resistant, S: Susceptible

having no chlorophyll, and also on the green parts of leaves of viridoalbina, having chlorophyll. The susceptibility of this mutant to leaf rust
was higher than that of normal plants. The mutant strain "basi-viridis
II" was susceptible to stem rust.

C.

CYTOLOGY AND GENETICS OF NICOTIANA ANDSOME OTHER PLANTS
25.

Cytogenetic Studies of the Genus Nicotiana XI(By YO T AKENAKA)

The reduction divisions in PMC's were studied in 3 interspecific hybrids:
N. knightiana x N. tabacum, 4x N. tabacum x N. benavidesii and 4x N.
tabacum X N. langsdorjfii.
1) F 1 of N. knightiana (n= 12) X N. tabacum (n=24).
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 0 to 5, withthe mode at O. The frequency of PMC's with one bivalent followed thatof PMC's with only univalents, and amounted to 30% of the total numberof cells observed. The frequencies of PMC's with 2 bivalents and 3
bivalents were 20% and 10% of the total number, respectively. PMC'swith 4 bivalents were very rare. Considering the small number of bivalents, it is difficult to determine whether the chromosomal affinities are•

RESEARCHES CARRIED OUT IN 1958

51

allosyndetic between the genomes of the parents or autosyndetic between
the two genomes of N. tabacum which is of amphidiploid origin.
2) F 1 of 4xN. tabacum (n=48)xN. benavidesii (n=12).
This hybrid was produced for the purpose of introducing a gene or genes
for immunity against the common mosaic disease from N. benavidesii into
cultivated tobacco.
At MI in PMC's of this hybrid, the total number of bivalents and trivalents per cell was 24. The number of trivalents ranged from 0 to 6,
with the mode at 2. PMC's with 5 or 6 trivalents were very rare.
At meiosis of the F 1 of 2x N. tabacum x N. benevidesii GOODSPEED (1954)
observed the range of chromosome pairs to be from 0 to 6, with the mode
at 3. Considering that in my study hybrid plants with two chromosome
sets of N. tabacum and one chromosome set of N. benevidesii have been
used, it may be said that the results of my observations are generally inagreement with GOODSPEED'S data, .because the affinity between homologous
chromosomes may in this hybrid be saturated by the presence of two
chromosome sets of N. tabacum.
3) F 1 of 4x N. tabacum (n=48)xN. langsdorffii (n=9).
The purpose of producing this hybrid was to introduce a gene or genes
for immunity to mildew and a gene or genes for resistance to black rootTOt from N. langsdorffii into cultivated tobacco.
At MI in PMC's of the F 1 of 4x N. tabacum x N. langsdorffii, the total
number of bivalents and trivalents per cell was 24. The number oftrivalents ranged from 0 to 5, with the mode at 1. PMC's with 4 or 5
trivalents were very rare. In my laboratory, Mr. Hu (1956) observed
meiosis of the hybrid between N. tabaC1tm and N. langsdorffii, and observeda range from 5 to 12 chromosome pairs with the mode at 11. Considering
his data, more trivalents than those observed by myself may be expectedin the F 1 of 4x N. tabacum x N. langsdorffii. But the small number oftrivalents found in this hybrid may be due to saturation of the affinity
between homobgous chromosomes by the two chromosome sets of N.
tabacum present in this hybrid.

26.

Cytological Studies in the Genus Euphorbia III(By Sh6hachi SHIMOYAMA)(1) Somatic chromosomes of Euphorbia Lathyris L.
Euphorbia Lathyris is a native of South Europe and Southwest Asia andit is cultivated in Japan as a medical plant.
The materials for the present study were supplied by the Botanical
Gardens of Technical University, Netherlands; the Botanical Gardens of

52

RESEARCHES CARRIED OUT IN 1958

Munchen, Germany; the Botanical Institute of the University of Liege,
Belgium and the Botanical Gardens of the University of Tokyo.
Somatic chromosomes in root-tip cells were observed by the acetic orceinsquash method. The chromosome number was without exception 2n=20,
in agreement with the observation of PERRY (1943). According to myobservations the chromosome set of this species consists of 18 V-shaped
chromosomes and two J-shaped chromosomes with a satellite on the shortarm. Tho karyotype can be expressed as follows:
K(2n)=20=2Am+2Bm+2 fCsf+10D"'+4E'" .
The basic chromosome numbers of Euphorbia are considered to be 6, 7,
8, 9 and 10 (PERRY 1943, TISCHLER 1950, DARLINGTON and WYLIE 1955). The
chromosomes of plants with the basic number 10, including E. Lathyris,
are usually larger than those of plants with other numbers.
(2) Karyotypes of Euphorbia Sieboldiana Morr. et Decne. and E. Sieboldiana var. idzuensis Nakai.
Euphorbia Sieboldiana is distributed in Southern Kurile, Sachalin(southern part), Hokkaid6, Honsyu, Sikoku, Kyusyu and Korea (?). This
species is very polymorphic and presents a troublesome problem for thetaxonomist.
E. Sieboldiana s. str., collected at Tazimagahara, Provo Musasi, has 20
somatic chromosomes; 14 with a median centromere, four with a submedian centromere and two with a subterminal centromere. The karyotype can be expressed as follows:
K(2n)=20=14Am+4B-,m+2Csf .

E. Sieboldiana var. idzuensis is endemic in Izu-Peninsula, Izu-Osima andProvo Kazusa. In this variety, differences in the karyotype between
habitats were found. The material obtained at Misima, Heda-misaki andIr6zaki in Provo Izu were similar to E. Sieboldiana in their karyotype, but
the plants collected at Izu-Osima had a satellite on the short arm of the
C-chromosomes. The karyotype of the latter is represented as follows:
K(2n) =20= 14A"'+4BS?"+2 tCst .
E. Sieboldiana includes various varieties other than idzuensis; miyazimensis Hurusawa, montanus Tatewaki, ohsumiensis Hurusawa and Shikokiana Hurusawa. They are now being studied from the cytogeneticalpoint of view.
(3) Chromosome variations in Euphorbia pekinensis Rupr. and allied
species.
Euphorbia pekinensis is perennial and widely distributed in Japan, Korea,
Manchuria and China. In Japan, it is grown in Honsyu, Sikoku andKyusyu. Typical localities of this species are found in North China.

•

53

RESEARCHES CARRIED OUT IN 1958

This form is very polymorphic and may be divided into many subspecies,
varieties and forms. Moreover, there are several species whose distinction
from E. pekinensis is sometimes obscure.
In a previous paper, chromosome numbers of 2n=28 and 56 were reported for this species; plants coming from mountainous regions had theformer chromosome number, while the latter number was found in specimens collected on fiat ground.
In 1958, one variety and one species closely allied to E. pekinensis were
examined cytologically.
E. pekinensis var. ibukiensis Hurusawa
2n=52
E. adenochlora Morr. et Decne.
2n=26
E. pekinensis var. ibukiensis occurs at a high elevation on Mt. Ibuki in
Provo Omi: the plants are low, herbaceous and fasciated. E. adenochlora
grows gregariously on wet land and is distributed in Hokkaido, Honsyfr
and Kyusyu.
Perry (1943) reported 2n=24 for E. pekinensis from material of unknown
origin. It seems that in this species-group of Euphorbia both polyploidy
and aneuploidy have been effective in speciation.
(4) Chromosome numbers of some Euphorbia species native to foreigncountries.
In 1958 the chromosomes of six species native to foreign countries were
examined. The results are given in Table 1, together with the sourcesof materials.
Table 1.
Speciesi-2nEuphorbia Peplus

16

E. bulbolina

20

E. cyparissias

20
20

E. stricta
E. marginata

SourceI----------

I

E. palustris

List of chromosome numbers.

Jardin Botanique de la Ville de Nancy, France;' Jardin Botanique de I'Universite et de la Ville,
Besan~on, France; Jardin Botanique de I'Universite de Lisboa, Portugal
I

20
i 56

Botanischer Garten der Universitat
Deutschland"

Gottingen,Hortus Botanicus Academiae Scientiarm RSS Tur, comaniae Ashkhabas USSR

"
"

54

RESEARCHES CARRIED OUT IN 1958

27.

Cytotaxonomic Studies in Veronica and Related Genera, I(By Takashi YAMAZAKI and Tuguo T ATEOKA)

As part of a taxonomic investigation, the chromosomes of Veronica aEd
related genera have been examined by the junior author. In 1958 the following results were obtained.
Veronicastrum sibiricum Pennell var. zuccarinii Hara
2n=34
V. sibiricum var. japonicum Hara
2n=68
Veronica schmidtiana Regel ex Fr. Schmidt var. bandaina
Makino form. senanensis Yamazaki
2n=34
V. routunda Nakai var. subintegra Yamazaki
2n=33*
V. kiusiana Furumi subsp. miyabei Yamazaki var. villosa
2n=34
YamazakiV. kiusiana subsp. maritima Yamazaki var. maritima
2n=68
Yamazaki
2n=68
V. ornata Monjuschko
2n=18
V. nipponica Makino ex Matsumura
2n=36
V. onoei Franch. et Savat.
2n=54
V. hederaefolia Linn.
2n=14
V. repens Clar.
2n=16
V. sp.**

28.

Development of Seeds and Embryos in Three Crosses between
Diploids and Artificial Tetraploids(By Kazuo FURUSATO)

In general, when artificially produced tetraploids are crossed with theoriginal diploids as pollen contributors, the seeds ripen and germinate well.
But the reciprocal crosses, diploid x tetraploid, usually give non-viable seeds
only. The development of seeds and embryos in the latter crosses has
been investigated in Nicotiana tabacum, Citrullus vulgaris and Citrus sp.
with the following results.

Nicotiana tabacum
At self-fertilization and in crosses between diploid strains approximately
1.

* This unexpected number may be derived through the loss of one chromosome
from 2n=34. The material used in this observation was only one individual.
** The seed sample of this specks wa· supplied by the Edinburgh Botanic Garden. According to a label attached to the sample, the species was obtained from
Tibet.

RESEARCHES CARRIED OUT IN 1958,

55

one day after pollination the male nucleus divides and two nuclei arefound in the pollen tube. After another day they enter the embryo-sac
and perform double fertilization. One or two days after fertilization, theegg cell starts to divide and in the course of the next 5-7 days the differentiation into cotyledons enclosing the pulmula and radicula is perfected.
From then on the further development takes its regular course, and about
20 days following pollination the seeds are ripe.
But 'when the pollen of a colchicme- produced tetraploid is dusted on
the stigma of the original diploid, the germination of the pollen grains is
considerably slower than in the reciprocal combination. Moreover some of
the grains show abnormal growth, and quite often twisted pollen tubes
can be seen. After fertilization the egg cell starts to divide but thisprocess is very slow if compared with the 2x X 2x-combination. No differentiation of the embryo can be detected. Even after 30 days only a cellmass of globular shape is found which finally shrinks away in the immature seed. At the same time, the endosperm nucleus undergoes a numberof divisions and in the first 4-5 days following fertilization free nuclei canbe observed. Their number is much smaller than when the pollen is
furnished by a diploid tobacco. It seems that poor development of theendosperm is one of the reasons for the poor growth of the embryo.

2. Citrullus vulgaris
When a diploid is pollinated by another diploid the seeds ripen well
25-30 days after pollination. But when a colchicine-produced tetraploidfurnishes the pollen dusted on a diploid stigma, the same phenomena
can be observed as in tobacco, and the cell mass representing the embryoremains undifferentiated. However, when the mother is the diploid Citrullus colocynthis and the pollen is furnished by the tetraploid C. vulgaris,the development of seed and embryo is normal and viable seeds areproduced. The cause of this exceptional behavior is not clear. Perhaps
the firm flesh of colocynthe protects the developing seeds from drying out.
3. Citrus sp.
In pollinations between diploids, seeds are easily produced, in mono- aswell as in polyembryonic varieties. But when the pollen derives froma tetraploid, the monoembryonic varieties produce aborted seeds only andthe fruits are practically seedless. In polyembryonic varieties a few small
nucellar embryos are developed and the seeds are undersized. The poor
growth of the nucellar embryos may be caused by the deficient development of the endosperm.

56

RESEARCHES CARRIED OUT IN 1958

29.

Frequency of 3x Seedlings in Several" triploid" Varieties of
Sugar Beets(By S. MATSUMURA, A. MOCHIZUKI and M. NEZU)

The somatic chromosome numbers of the offspring were determined in
the best combination, No. 4398 (4x) X No. 162 (2x). VVhen 2x and 4x seeds
were sown mixed in the ratio of 1 : 5, the frequency of 3x seedlings was
the highest, namely 46.5% (Ann. Rep. No.7, p. 49). In order to produce
Table 1.
=--~---

Frequency of 2x, 3x and 4x obtained from European" triploid" varieties.
-----=---......_-------==---=-.'='---,-'-==-.-- -Variety

2x

3x

4x

Total

._-----

Hilleshog-St· poly
(Sweden)
Total
%

49
14

43
18

7
13*

99
45

63
43.75

61
42.36

20
13.89

144
100

44
9

43
19

14
8

101
36

53
38.69

62
45.25

22
16.06

137
100

73
5

100
13

32
7*

205
25

78
33.91

113
49.13

39
16.96

230
100

27
0

40
37*

6
6

73
43

27
23.28

77
66.38

12
10.34

116
100

10
20.00

40
80.00

0
0

50
100

26
0

74
2

28
14

128
16

26
18.05

76
52.78

42
29.17

144
100

27
4*

35
11*

5
3*

67
18

31
36.47

46
54.12

8
9.41

85
100

------_.. _ - - -Hilleshog.R·poly(Sweden)
Total%

KL-Cercopoly(Germany)
Total%

Polyrave(Netherlands)----------

Total
%

Trirave(Netherlands)-------------_.

Beta-I(Hungary)
Total%----,------- - -'--,------------_._~---------

Beta-3(Hungary)
Total%* inc!. heteroploid•

RESEARCHES CARRIED OUT IN 1958•

57

" triploid" seeds on a commercial scale, promotion of the percentage of 3x
plants should be attempted. Recently" triploid" varieties have beenbrought to market by Sweden, Germany, Denmark, Netherlands andHungary. Table 1 shows the frequencies of 2x, 3x and 4x seedlings inseveral European "triploid" varieties. There is no significant differencein the percentage of 3x plants between our combination, No. 4398 X No. 162,
and the Swedish or German varieties. The Dutch varieties show the best
results. A new Dutch triploid variety, "Trirave ", has been advertised
recently as a triploid obtained by using 2x male sterile plants. Our experiments show beyond doubt that in sugar beets 3x seeds can be obtained
through natural pollination even from 2x mother plants, when 2x and 4x
beets are planted. If 2x mother plants are male sterile, almost all seeds
obtained from them must be 3x.
In another experiment, attempts have been made to induce tetraploidy
by colchicine treatment in several resistant American varieties (GW 602,
GW 674 etc.) so as to use them as tetraploid parents.

30.

Heterostyly and Pollen Grain Number in Buckwheat(By Yukio DOIDA)

According to the number of pollen grains formed in a pollen sac, five
types of plants can be distinguished in Polygonum. They are: pollen sacs
with (1) 8, (2) 16, (3) 32, (4) 128 and (5) 256 pollen grains (DorDA, 1957).
In 1958, the pollen grain formation of long-styled flowers of buckwheat
was examined and compared with that of short-styled flowers.
It was found that both long-styled and short-styled flowers have different
average numbers of pollen grains formed in a pollen sac (Table 1); themode for the long-styled flowers was at 48 while that for the short-styled
was at 32. Moreover, the variance for the long-styled flowers was clearly
Table 1.

Number of pollen grains in a pollen sac (P. fagopyrum).
Number of pollen grains
less
than

23

co

Short-styled
flowersooZLong-styled
flowers

til

(J.J

til

-

32

U

10,31

I

48

RESEARCHES CARRIED OUT IN 1958

58

larger than for the short-styled flowers.
It is well known that in plants with heterostyly the size of the pollen
grains is different in long- and short-styled flowers. This is true of buckwheat too. Moreover, the long- and short-styled flowers of this species
are characterized by a different average number of pollen grains, as Table
1 shows.

31.
1.

Developmental Studies in the Genus Polygonum
Microsporogenesis in Polygonum persh'aria(By Yukio DOIDA)The number of pollen grains per pollen sac is a constant trait in Polygonum species. In a previous paper (DoIDA, 1957), the author distinguishedfive types, based on different numbers of pollen grains per pollen sac. It
was found in 1958 that P. persicaria does not fall into any of the five
types. Usually this species has only four pollen grains in a pollen sac.
The development of the pollen mother cell starts with the differentiation of a large cell from the hypodermal cell layer of the anther primOl-dium. This cell is soon clearly distinguishable by its size from the surrounding cells. It continues to grow and increase in size but does notundergo mitotic division and becomes in this condition a pollen mothercell. On the other hand, the cells surrounding the pollen mother cel!
divide and form four cell layers. Regular meiotic division takes place inthe pollen mother cell, and a tetrad and subsequently four pollen grains
are produced. Following the meiotic stage, the inner two of the four cell
layers surrounding the pollen mother cell begin to degenerate. When ananther reaches maturation, its peripheral zone consists of two cell layers.
Rarely a mitotic nuclear division of the sporogenous cell takes place andeight pollen grains are formed in a pollen sac. Dwarf-pollen grains are
seldom found. Therefore it is assumed that meiosis is very regular. This
assumption was confirmed by direct observation. Pollen sacs having nopollen grains are also found very rarely. It was histologically confirmedthat in such cases the sporogenous cell usually differentiates but the periTable 1.

Number of pollen grains in a pollen sac (P. persicaria).
No. of pollen grains
4

No. o~sesI

8·-1- 24~ J_2~~

12

Total

Mode

269

4

3+2m*

- - -

I

* m: micro-pollenoRESEARCHES CARRIED OUT IN 1958

59

pheral zone of the anther, especially the tapetal cells, do not develop
satisfactorily. The degeneration of the pollen mother cell may be caused
by insufficient supply of nutrition which ensues from insufficient development of tapetal tissue.

,II.

32. Developmental Studies in the Genus Polygonum
EjJects of Gibberelline on Microsporogenesis of Polygonum fagopyruJ1z(By Yukio DOIDA)The author has examined the action of gibberelline upon the development of microspores in Polygonum fagopyrum. This material was chosenbecause of the small number of pollen grains formed in a pollen sac. The
concentrations of gibberelline used were 0-100 p.p.m.
The results are as follows:(1) The higher the concentration of gibberelline, the smaller is thenumber of pollen grains per sac. At concentrations higher than 10 p.p.m.
pollen formation was markedly inhibited, as Fig. 1 shows.
Short-styled flower
Long-styled flower

(/)

C

40

'0:;

•••• -0-•••••• ' \ \... u
b1J (/)
ell

C

~

,

~

a:i 30

0:':::.....""8.
o ro

20;:; .S1110.s§v::;:;;

1

1L.-~_~_~_~~_....,......._
o

0.1

1.0

10.0

25",0

50.0

100.0

Cone. of gibberelline (p.p.m.)
Fig. 1(2) The diameter of pollen grains remained unchanged but the anthers
became shorter with increasing concentrations.
(3) The surface area of the epithelial cells was considerably reduced
after treatment with 50 p.p.m. in comparison with the control. The surface area of the epithelial cells was 1.58+0.19 in the control and 1.236±
0.189 after gibberelline treatment. The figures represent relative values
obtained from planimeter measurements. The difference was significant(P<0.05).

60

RESEARCHES CARRIED OUT IN 1958

From the above results it is clear that gibberelline had an inhibitory action on the microsporogenous tissue of Fagopyrum. The same tendency
was observed in some flower organs.

33.

Further Studies on Cherry-red Leaf in Tobacco(By Kan-Ichi SAKAI and Shin-ya IYAMA)The writers have formerly reported that in the "Bright Yellow" tabacco
variety, strains markdly differ in the grade of expression of cherry-red
leaf color, and that genes for high cherry-redness might be dominant, as
the results of investigations of F 1 and F 2 plants have shown. This year,
F g lines, derived from one of the already reported crosses between" Bright
Yellow" strains, were observed. As in the years before, in order to obtain a formula which gives proper weightings to the index-numbers forthe grades of cherry-redness, 18 different strains were grown in addition to
the F g lines. The F g test comprised plots of 22 F g lines, parental strains
and F1's of reciprocal cross combinations, each consisting of a row of 15
plants. The plots were randomized with three replications. The 18 strains
were grown in rows of 12 plants, with two replications.
At maturity, the five upper leaves were harvested and after having been
dried, were graded into five classes with the index-numbers 0 to 4. Then,the average cherry-redness of each plant was calculated from the data byusing the weighting formula.
1) Test of strains: From the data of the strain test, the following
formula was obtained maximizing the ratio of variance among plants tothat within a plant, i.e.,

where XO to X4 are the number of leaves falling in the respective classes,
and X represents the average grade of cherry-redness of a plant. Varianceanalysis of the score X showed that the variation among strain means
was significant. Having computed from the results the variance components
for individual plants and for strains, the heritability value was found tobe as high as 0.50 for a plant, and 0.89 for a strain.
Further, variance analysis was made of the data for the four years,
1955-1958. The results showed, as given in Table 1, that the variation
among strains was highly significant, and the interaction between strain
and year was insignificant. It may be concluded, therefore, that the gradeof cherry-redness is essentially a genotypically controlled character.
2) Test of F g lines: Variance analysis of the data showed that the••

RESEARCHES CARRIED OUT IN 1958
Table 1.

It

61

Variance analysis of the data of strain test
for cherry-redness.

Variation
due tod.f.

Mean square

Year
StrainStrain x Year
Error

3
17
51
90

.0681*.2488**
.0220
.0159

* Between 5% and 1% levels.
**

Above 1 % level of significance.

differences between lines were significant, as given in Table 2. It wasalso found that the correlation between the F 2 measurements and the Fa
line means was as high as 0.727. Besides the high heritability value as
already given, this may also indicate that selection for this character
would be successful. However, the number of effective genes could not
be estimated.
Table 2.

Variance analysis of the data of Fa test.
--

Variation
due tod.f.

Replication
Line
Error

1
21
42

I

Mean squareI.1228**.1277**.0095** Above 1% level of significance.

•3) Relation between cherry-redness and quality of leaves: An insignificant correlation coefficient r=0.234 was found between the grade ofcherry-redness and the quality of leaves.

34.

Sexuality in Rumex hastates(By Y6 TAKENAKA)

Rumex hastatus is a gynodioecious species consisting of females and androhermaphroditic plants. The latter flower have 5 perfect anthers and anundergrown pistil.
In the offspring of the females, 79-9- and SOet) were found. From thepistil of the androhermaphroditic plants, 35-9- and 92et) were obtained. If
we designate the gene effecting male expression M (" Realisator "), thegenotypes would be mm for the females and Mm for the androhermaph-

62

RESEARCHES CARRIED OUT IN 1958

roditic plants. The cross 111m X Mm should give females and males in the
proportion of 1: 1, as it was observed to do. In the offspring of Mm weshould have Imm: 2Mm: IMM, i.e. 1¥: 3'2';, a proportion which was actuallyfound. One third of the males, those which are MNI, should be puremales. Moreover, from crosses between those pure males and the females
we should expect 100% androhermaphroditic males, l'vlrn.
A detailed investigation of this interesting problem is under way.

D.

BIOMETRICAL GENETICS AND PROBLEMSOF BREEDING
35. Inheritance of Competitive Ability in Rice
(By Hiko·Ichi OKA and Kan-Ichi SAKAI)

It was demonstrated early in this series of competition studies that thecompetitive ability of a strain is a genetically determined character. It is
known that varieties of the Indica type of rice generally show higher
competitive ability than those of the Japonica type (Annual Report No.6:
83-84). This report deals with the inheritance of competitive ability observedin F 2 and F 3 lines of a cross between two rice varieties, Kinoshita-mochi(Japonica) and U-koh (Indica, from Taiwan). In this cross, though theparental varieties were distantly related, the F 1 was almost completely
fertile in both pollen and seed setting.
Design of experiment: Plots, each consisting of a row of 15 plants, belonging to the parental varieties, F 2 and 40 F 3 lines were randomized withfour replications. The interval between rows was as large as 50 cm so as
Table 1.

Variance components for the increments or decrements in a tester
strain due to competition with hybrid or parental plants ofa cross between two rice varieties.
Panicle number

Plant weight1--------- - - - - - - - - - - - --Observed

Expected

Observed

Expected

V F2

12.47

VP3
V F3
E1
E2
D
H

12.57
3.20
11. 7911.01
1. 70
2.96±1.065
0.33±2.922

347.3

3.20
12.02
10.90
1.72

348.8
30.8
341.6
334.4
20.6
17.7±14.57
22.1±40.0l--------_...:..._------- - - - - - - - -

30.8
344.7
332.9
21.2

RESEARCHES CARRIED OUT IN 195863

to avoid competition between adjacent rows. In each row, the plants werespaced at 25 em, and between them, two plants of a "tester" strain wereinserted, reducing the distance between plants to about 8 em. As a tester,
Yaeho, a rice variety common in Sizuoka Prefecture was used. It was
assumed that the two tester plants standing on each side of a plant belonging to the hybrid or parental population would show the effect ofcompetition. The sums of the measurements of each pair of tester plants
were recorded and used for variance analysis. Panicle number and plantweight were the characters investigated.
Results: Partitioning of variance components was made by MATHER'S
method. The results are given in Table 1.
The data in the table show that the inheritance of competitive ability
can be analysed by the same method as used for ordinary quantitative
characters, if the experiment is designed as in the present study. Thissuggests that competitive ability is controlled by Mendelian factors. However, the number of effective factors could not be estimated because thevariance in the F 3 lines ,vas too great.

36. Effect of Soil Fertility on Competitive Ability in Upland
Rice Varieties(By Kan-Ichi SAKAI and Shin-ya lYAMA)
It has been shown before by OKA and SAKAI (Annual Report No.6) thatcompetitive ability in lowland rice varieties is affected by soil fertility andother environmental conditions. In this paper, the results of an experimenton the effect of fertilizers on competitive ability of an upland rice variety
are reported. Competitive ability (P) was computed based on the change
in the proportion of genotypes after one generation of mix-planting, byusing the formula (presented by the senior writer in 1954),pal-ao+(l-al)aoq
ao(l-ao)

A Japanese upland rice variety, Norin-mochi No.1, and the so-called.. Red Rice" (an Indica-type rice occurring in upland rice farms in somelocalities in Japan, which is strong in competition with ordinary uplandvarieties) were used. Seeds of these two strains were mix-sown, the initial
proportion of "Red Rice" (ao) being 5%. The experimental plots had fivefertilizer levels, i.e., 4, 1, 1/2, and 1/4 of the normal dosage and a nonfertilized plot. The normal dose consisted of N 6.0 kg/ha, P Z0 5 7.5 kg/ha
and K 20 3.75 kg/ha of ammonium sulphate, calcium superphosphate andpotassium sulphate.

RESEARCHES CARRIED OUT IN 195864

At the same time, plots of each variety were raised. From the numberof seeds harvested from these plots, the propagation rates (1-q) of "RedRice" relative to Norin-mochi No. 1 at different fertilizer levels were
estimated, as given in Table 1. Competitive ability (p) at each fertilizer
level was computed from the proportion of "Red Rice" seeds in the mixplanted plots (al), as also given in Table 1.
Table 1. Relative propagation rate (1- q), proportion of seeds in mix-plantedplots (al) and competitive ability (p) for" Red Rice". (ao=0.05)
Fertilizer
dose

1-q

al

aI, Expected

Observed

without
competition

p

0.035
0.077
0.105
0.130
0.149

0.038
0.044
0.046
0.046
0.056

-0.063
0.683
1.243
1.766
1.966

----

4
1
1/2
1/4
0

0.746
0.870
0.907
0.908
1.123

~-----~-The data in the table show that when four times as much fertilizer as
the standard dose was applied, "Red Rice" was no longer a strong
competitor against Norin-mochi No.1, but the competitive ability of"Red Rice " tended to increase with the decrease of fertilizer. Thissuggests that the propagation of "Red Rice" mix-growing in upland rice
farms would be promoted by poor soil fertility.

37.

Studies on the Breeding Behavior of Wild Rice(Oryza rufipogon and Oryza perennis)*(By Kan-Ichi SAKAI and Takashi NARISE)

A biometrical method for the estimation of the percentage of outcrossing
in plants by using polygenic characters has been reported by SAKAI and
IYAMA (1957). In the present study, this method was adopted for two
species of wild rice, O. rufipogon (0. sativa spontanea) and O. perennis.
The habitats of the material were as follows:
Species
Notation
LocationO. rufipogon
Chi
Chinsurah, West Bengal, India
III
Illuppaiyadichchenai, East coast of Ceylon
PP
Pottuvil Periphery, East coast of Ceylon
PT
Pottuvil Town, East coast of Ceylon
O. perennis
Vey
Veyangoda, West inland of Ceylon
Yag
Yagoda, West inland of Ceylon---------* This work was supported by Grant RF 57080 from the Rockefeller Foundation.

RESEARCHES CARRIED OUT IN 1958..

65

Seeds were collected at random in wild populations on individual plantbasis. Young plants grown in pots for about one month were transplanted
into the field at Peradeniya, Ceylon on the 24th of June, 1958. After
harvesting, the width of spikelets was measured. With regard to detailsof the estimation, the reader is referred to the 7th issue of the Annual
Report (page 53).
The analysis of variance, expected components of mean squares andpercentage of outcrossing are given in Table 1.
Table 1.
Mean square

Source ofvariationI

Between strains
Between individuals
within strain
Within individual
within strainPP
PT 1 Vey
Yag----I-------- . - -.. - - - ---

.,

--

.0325

.12281.0005.0046, .0065--- ----.0-04-6
.0005

(J'o~

.,
a']J-

:

.1210 1 .3184
1.39781

_~._

...

_-

.00571 .0062.00621 ere2
1.00651-~~0571 .00621 .0062

.0118

.0115

.03131 .0392 i .0180

.0046

.0554

.0652

.39781- - - - - -

.

1

1

.11921.0489:---'----

I
I.1864ere2+klero2.0032--.---

Percentage of outcrossing (%)- _ . _ - - - _ ..

0.1914 0.309310.304112.0100 1.2509 0.6467 ere2+klero2+k2erlJ2

1

a'e~

Expecta tion ofmean square

50.67

20.00

31.60 1 7.41

1

26 . 51 1 22 .421.-.-.------

Table 1 shows that percentages of outcrossing in O. rufipogon were on anaverage 25-3096 although they varied from population to population, i.e.
from 50% in Chi to 7% in PT population. Similar values were also foundin two populations of O. perennis.
From these results, we may be right to conclude that wild rice plants,
Oryza rufiPogon, growing in their native habitats are partially allogamous,the percentage of outcrossing being roughly estimated as 30%. The
percentage of outcrossing may not be the same among different populations.

66

RESEARCHES CARRIED OUT IN 1958

38.

Genetic Variability in Wild Populations of Oryza perennis
and O. ruJipogon*(By Kan-Ichi SAKAI and Takashi NARISE)

Analysis of genetic variability in wild populations of Oryza perennis andO. ruJipogon (0. sativa spontanea) was carried out in 1958 with materials
collected from different spots in India and Ceylon. Regarding the methodof investigation, reference should be made to the last issue, No.7, of thisAnnual Report (page 67). Localities where collection was made are listed
below:
Notationofpopulation
Oryza perennis CH
JA
CT-A
CT-B
CT-C
SpeciesBG
PH
SM
O. ruJipogon

CH
CN
JA

F-b
PH
T-1
T-4
T-5
TR
Pt

SD
LT

No. ofindividuals
collected
Chinsurah, West Bengal, India
50
Jagatapur, Orissa, India
30
Cuttack, Orissa, India
50
Cuttack, Orissa, India
50
Cuttack, Orissa, India
50
Baliguda, Orissa, India
50
Phagad, Orissa, India
30
Samalkot, Andhra, India
50
Chinsurah, West Bengal, India
21
Canning, West Bengal, India
50
Jagatapur, Orissa, India
40
Cuttack, Orissa, India
58
Phagad, Orissa, India
36
Samalkot, Andhra, India
75
Samalkot, Andhra, India
75
Samalkot, Andhra, India
72
Trichur, Kerala, India
50
Puttalam, West coast of Ceylon
24
Sadulgona, East coast of Ceylon
50
Laphugala Tank, East coast of Ceylon
41
Locality

Analysis of variance for length and width of spikelets is presented inTable 1, and the mean values for those characters are shown in Table 2.
The data in Table 1 show that the variations between species, between
populations of the same species, and between individuals within a population are all statistically significant. In Table 2, we find that O. ruJipogonhas, in general, slightly longer and wider spikelets than O. perennis.

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

RESEARCHES CARRIED OUT IN 1958
Table I.

67

Analysis of variance of spikelet length and width in wild
rice populations.
Mean square

Source of variationd.f.

Between species
Between populations within species
Between individuals within populations
Within individuals

1
18
932
8,568

Spikelet
lengthSpikeletwidth

284.836**
26.527**1.880**
0.216

63.013*9.236**
0.269**
0.012* Significant at the 5% level.
** Significant at the 1% level.
Table 2.

Mean value of spikelet length and width in wild rice populations.

SpeciesPopulation

Spikelet length

Spikelet width

CH

PH
SM

8.04 mm ±0.267
7.64±0.4288.36±0.3628.10±0.2027.86±0.6208.17±0.4027.95±0.3187.88±0.369Mean

8.~~_±O.248 __ 1-2.SS--~0.16_2 _ _- - - - - ~ - _ _-2.39 mm ±0.106
..

JA
CT-A
CT-B
CT-C

o. perennis

BG__ ~_lI

I±0.025±0.191±0.086±0.250±0.265±0.173±0.255

LT

8.59
8.40
8.24
7.75
8.30
8.50
8.60
8.81
8.47
8.42
8.02
8.43

±0.287±0.360±0.409±0.251±0.298±0.102±0.352±0.299±0.365±0.330±0.296±0.450

2.70
2.79
2.77
2.63
2.82
2.77
2.86
2.84
2.75
2.63
2.71
2.46

±0.150±0.071±0.1l6±0.1l6±0.084±0.091±0.069±0.102±0.159±0.070±0.200±0.146Mean

8.38±0.2672.73±0.121

CH

CN
JA

F-b
PH

O. rufipogon

I

2.75
2.51
2.79
2.59
2.75
2.58
2.33

T-1
T-4

T-5
TR
Pt
SD

68

RESEARCHES CARRIED OUT IN 1958

Populations of O. ruJipogon collected in India were investigated to findwhether or not the length and width of spikelets differ according to locality.
No geographical cline was found, however.
Further, the magnitude of genetic variability in these wild rice species
as shown by the heritability values of spikelet length and width wasinvestigated. The values of heritability (the amount of genetic variancein percent of total variance) thus found are presented in Table 3.
Table 3.

Heritability values in wild populations of two Oryza species.
Heritability value

SpeciesPopulation
CH

O. perenn:is

JA
CT-A
CT-B
CT-C
BG
PH
SM
CH
CN

O. rufipogon

JA
F-b
PH
T-1
T-4
T-5
TR
Pt
SD
LT

Spikelet length

Spikelet width

0.7536.3312.5299

0.7580
.1310
.1037
.4098
.8048

.5828
.6160
.5314
.1386
.5536
.4419
.5813
.1824
.4488
.2823
.6249
.9466
.9780
.7981
.2660
.6041
.7231

.9124
.3630
.9929
.9375
.3530
.6149
.6176
.2485
.5837
.6227
.5688
.4945
.4454
.5710
.9328

Fig. 1 shows the geographical distribution of heritability values for the
average of the values for the two characters. It is noticeable in Table 3that wild rice populations are generally highly heterogeneous. It seems
that populations collected from Orissa, India, have lower heritability values
though they may differ according to localities.
Genetic as well as environmental correlations between spikelet length andwidth are shown in Table 4.

69

RESEARCHES CARRIED OUT IN 1958

1~~~'~~~lh .,.,.1·.,/:e.""!l.

Cuttack (0.2"11)(g:~~6~)

Fig. 1. Geographical distribution of the average heritability values of spikelet
length and width.
Table 4.
Specieso. perennis

Environmental (r") and genetic (rg) correlations between
spikelet length and width.
Population

CH
JA
CT-A
CT-B
CT-C

BG
PH
SM
CH

CN
JA
F-b

PH
O. rufipogon

T-1

T-4

T-5

TR
Pt

SD
LT

rg

0.1477
.0116
.0914
.1864
.0155
.3979
.0577
.1175

0.0549
.5415
.4787
.6887
.8410
.3080
- .2027
.2079

.2896
.8409
.0108
.0030
.0133
.1274
- .7568
.0550
.1166
.0260
.4359
.1916

.1928
- .0335
.6634
.0688
.4973
.3378
.3733
.3307
.5441
- .2310
.5104
.3988

70

RESEARCHES CARRIED OUT IN 1958

Table 4 shows that the environmental correlation between the two
characters is generally insignificant except for a few populations, while
genetic correlations vary from population to population.

39.

Variation in Anthesis of Wild Rice Populations(Oryza rujipogon and Oryza perennis)*(By Kan-Ichl SAKAI and Takashi NARISE)

Progeny lines of plants collected from three populations of O. rujipogon(0. sativa spontanea) and two of O. perennis were investigated withregard to the date of heading.
lected are as follows:

The places where this material was col-Notation of
population
O. rujipogon
III
Illuppaiyadichchenai, East coast of Ceylon
PT
Pottuvil Town, East coast of Ceylon
PP
Pottuvil Periphery, East coast of Ceylon
Vey
Veyangoda, West inland of Ceylon
O. perennis
Yag
Yagoda, West inland of Ceylon
The progeny lines were sown in pots and after four weeks transplanted
to an experimental field on June 24, 1958. The mean number of days perline from sowing to flowering is presented in Table 1.
Species

Table 1.

Frequency distribution of number of days from 50wing to flowering in five
populations of wild rice.
No. of days from sowing to flowering

SpeciesPopulation

No.
of
lines

121

I

131 i 141I

I

130 I 1io I 150,---------

151

I

"
"

O. perennis

"

_.---

III
PT
pp

15
10

Vey
Yag

22
24

8

2

2
0

4
7
4

171

181

191

I

i

I

I

160 I 170

180

Average

190 I 200

I----

O. rufipogen

i

161

8
1
4
v

'0

11

1

8

7
24

-------,

150.88149.00141.02177.73182.62In Table 1, we find that the anthesis of O. rujipogon occurs markedly
earlier than that of O. peremzis. The results of analysis of variance andestimates of components of variances are presented in Table 2.

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

RESEARCHES CARRIED OUT IN 1958
Table 2.

71

Analysis of variance of days from sowing to heading in progeny
lines of five wild rice populations.

Variation due toSpeciesPolulations within species
Strain,; within population
Plants within straind.£.

Mean square

1

121,897.34**
2,323.63**
244.35**
98.26

3
84

510

608.02
21.93
21.59
98.26

Expectation of m.s.

cr tJJ2 -i-k1r.f 8'2 ~1-1~2rr p2
cr w 2 -j-k 1 cr s'2

6.462498.8147
196.6611** Above 1 % level of significance.
In Table 2, 0" A 2 represents the component of variance between species,
0"1'2 that bet,veen populations within species, O"S" that between strains within
populations, and O"w 2 that between individuals within a strain. By using
the values of components, the magnitudes of variation in heading date areshown in terms of intraclass correlation, and those between populations andbetween species are compared with each other. They are as follows:
Variability between strains within populations:_ 0 " / . =0.2198

l1 w 2+l.1 s'2

Variabiiity between populations within species:
(Jp'2

r

,.....

--.,--.,--., =0. 1041
O".,,'+O"s' +0"1"

Variability between species:

These values show that so far as anthesis is concerned, interspecificvariability was the highest, within each species, however, neither interpopulation nor especially interstrain (w'ithin the same population) variability
was negligible, which indicates that wild populations are genetically heterogeneous with respect to anthesis.

72

RESEARCHES CARRIED OUT IN 1958

40.

Genetic Studies on Seed Size in Rice Hybrids*(By Kan-Ichi SAKAI and M. E. R. PINTO**)The present paper deals with the inheritance of length and width of
paddy-grown rice seeds from six F 2 populations. The parental varieties
and the number of plants examined are as follows:
Cross
No.

Female parent

Male parent

Number of plants
investigated1

Podiwi B-8
Ptb-16
Bengawan B-27
Murungakayan 3
Ptb-16
N-48

Mas M-24
Mas M-24

136

2

3
4
5
6

137
142

Mawi B-11
Mawi B-11
SigadisMas M-24

129
90
141

Ten seeds from each plant were collected at random and their length
and width were measured. Analysis of variance of the data obtained wasmade and the within-individual and between-individual variance components
were estimated. The former component was taken as the estimate ofenvironmental variance, and genetIc variance was estimated from thebetween-individual variance component. Environmental and genetic covariance components were estimated in a similar way.
Heritability and genetic as well as environmental correlation computedfor the six F 2 populations are presented in Table 1.
Table 1. Heritability (h 2) and genetic (rG) and environmental (rE) correlationsof length and width of seeds in six F 2 populations of rice hybrids.
~ ~ - - - -Cross number

1

2

3

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

h2

SSeed length I

0.9092

1Seed width

0.4711

I

Width

rf!

5

0.7556

0.46150.06930.60820.37140.59270.61600.08450.44490.0394------------0.09640.1792I

6----------

0.6819I------

Lenxgth { rG4--------------

0.18260.1107-0.12180.0186

0.24200.32800.15930.49330.2351------* This investigation was carried out in Division of Botany, Department of Agriculture, Peradeniya, Ceylon.
** Agricultural Instructor, Department of Agriculture, Ceylon.

~(ESERACHES73CARRIED OUT IN 1958

From Table 1, we find that: (1) heritability values for seed length aswell as width are on an average as high as around 50%, those for length
being generally higher than those for width. (2) Among parental varieties
used in the crosses, Ptb-16, Sigadis and Mas M-24 are very similar in
seed length and v"idth. Very low heritability values obtained for bothcharacters in Ptb---16 x Sigadis (cross No.5) and rather high ones in the
cross Ptb-16xMas M-24 (cross No.2) suggest that Ptb--16 and Sigadis
must be very similar in their genetic make-up, while Mas M-24 must be
genetically different from Ptb--16 , although phenotypically they appear
alike. (3) The genetic correlation between seed length and seed width isfound to be as low as 0 or 10-2096 in the plus or minus direction, while
the environmental correlation between these two characters is rather highand always in the plus direction.
A number of useful suggestions have been deduced from the result of
this experiment.
41.

Inheritance of Size and Shape of Grains in Rice(By Shin-ya IYAlHA, Hiroko MORISHI;,\IA and Hiko-Ichi OKA)

The size of rice grains is known to be a character affected relatively
slightly by environmental agents. In view of its usefulness for a surveyof genetic variability in rice populations, its inheritance in two varietal
crosses was investigated by statistical-genetic methods in order to obtainfundamental knowledge for population studies. The two crosses used are
Kinoshita-mochi (Japonica) x U-koh (Indica, from Formosa), and Norin-mochi
No. 18 (Japonica, upland) x" Red Rice" (Indica).
The parental varieties and the F 2 and F 3 lines of each cross were grownin the experimental field of this Institute, according to a randomized block
Table 1.

Variances and covariances of grain length, observed aml
expected in Kinoshita-mochi x U-koh.
Observed

Population---_._--

II

VP2=1I2D-t-V1H-t-E1
VF3=1/2D-t-lhoH-t-E2
WF2/F3=1/2D-t-l/sH
VF3=1/4D-t-l/sH-t-El
E1

E2

.0856
.0744
.0434
.0731
.0381
.0392

.0871
.0699
.0425
.0646
.0318
.0091

Expected

Mean
.0864
.0722
.0430
.0689
.0349
.0241

.0882
.0702
.0459
.0634
.0386
.0261

74

RESEARCHES CARRIED OUT IN 1958

design with two replications. The unhulled grain length and width of 10
seeds were measured in each plant. Variances in the F 2 and other populations, and the covariance between the F 2 and F 3 lines were calculated
for length, width and length-width ratio, as shown in Table 1. The components of variances were partitioned by MATHER'S method. The results
are given in Table 2.
Table 2.

Character

Additive genetic variance (D), non-fixable genetic variance (H)and environmental variances (El , E 2 ) of grain characters.
Cross 1: Kinoshita-mochi xU-leah
Cross 2: Norin-mochi No. 18 x" Red Rice"
rJl
rJl

o

Length

g

Width

r1
12

LthjWidth

g

i

D

Ii

.0845 ± .0207.1388~= .0367.0204 ± .0095.0379± .0138.0197:1:: .0018.0177± .0080.0294± .0662.1013± .1179.0386± .0056.0220± .0111.0203±.0304.0042± .0443.0107±.0026 .0046±.0026.0110± .0038, .0048± .0038.0129 ± .0056.0535± .0256.0035 ± .0005.0071± .0022'"__'-____________ I U______

I.0261± .0057.0180± .0101

Amongvars.*
.5926
.1254

'

.0030 ± .0005 .1895.0061± .0022 I
1I* Variances among more than 600 varieties from various Asian countries.

As the data in Table 2 show, the observed variances gave a good fit
with the expected ones in cross 1 (standard deviations are relatively small),but in cross 2, deviations between observed and expected variances werelarger. The results of scaling tests proved that a convenient scale (inmillimeters) could be used in both crosses. It seems that the larger deviations in cross 2 might be due to changes in the segregation ratio caused
by gametic selection, because the F 1 plants were almost completely fertilein cross 1, but showed semisterility (pollen fertility being about 4096) incross 2.
It is found in Table 2, further, that the additive genetic variances dueto the crosses were one tenth to one fifth of the variance found amonga large collection or varieties from various Asian countries.
In the process of computation, a slight effect of linkage among relevent
genes was pointed out for the length-width ratio in cross 1, and for grain
length in cross 2. The number of effective genes could not be successfully
estimated.
The variances of grain characters within the same panicle were derived
from the data, in addition to the above computation. Based on thosevalues, components of environmental variances due to grains (6~1')' plants(6~1) and polts (6~lot) were computed. The results are given in Table 3,
together \'lith heritability values, the ratio of HID and other values

RESEARCHES CARRIED OUT I"'" 1958
Table 3.

75

Heritability value. ratio of HID, and comp~ments of environmental
variances found from the data.
Envircnmental va:'iance

Character ! Cross

HID

10E,/E1

!

0

<1~r

!
..

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

Length

{~

.348
.730

Width

{~

.995
.111

6.76
8.18
4.30
4.36

1

.655
3.023

8.57
8.59

Lth/Width

2

.0538
.1217
.0119
.0931

I
I

I

2
iT pi

rJ'~IO:

.0332
.0099

.0232
.0158
.0034
.0037

.0095
.0017

derived from the above computation. Th2 data in Table 3 shmv thatsignificant environmental variation in grain size due to the effects of plants
and plots occurs, in addition to the variation within a plant; in otherwords, the environmental variances found \vithin a panicle, between plants
and between plots are not of the same order. This should be consideredwhen we deal with genetic variations of this character.

42.

Polygenic Mutation, Induced by X-rays, in Biometrical Charactersof Drosophila melanogaster*(By Yukio YAMADA and Osamu KITAGAWA)

The dominant marked multi-chromosomal inversion technique which was
proposed originally to establish iso-homozygous lines was used in thisstudy, with the exception of the fourth chromosome.
We treated each sample of five males, taken at random from an isohomozygous line extracted by the above technique from a long inbred line,
as follows: (A) control, which was not irradiated, (B) 2000 rand (C) 4000 rof X-rays; 36, 47 and 30 homozygous lines were obtained, respectively.
In each line 40 females and 40 males were scored with respect to thenumber of abdominal (4th and 5th sternites) and sternopleural (right andleft sides) micro-hairs. This score, pooled for females and males, wasused for the analysis. The results obtained so far are given in Table 1.
As the table shows, the means of three treatments agreed well \vitheach other for both abdominals and sternopleurals. However, variancesamong the lines within treatments increased considerably in both Band C, as
compared with the control, A. Variance in A could possibly result fromspontaneous mutation and rarely occurring recombinations during isogenisation, and/or sampling by chance, but it could still be used to test the* This work was supported by Grant RF 57178 from the Rockefeller Foundation.

76

RESEARCHES CARRIED OUT IN 1958
Table 1.

Treatments~;.

Control
B. 2000 r
C. 4000 r

l'vIean and variance of bristle numbers.
Abdominals

Sternopleurals

No. ofsperm
sampled

J\Iean'lariancel\IeanVariance36
4730

31.290331.1801
31.1736

0.271378
0.924382**
0.940674**

15.188115.1165
14.9338

0.439861
0.903444*
0.374808* Significantly different frmn control at 5% level.
** Significantly different from control at 1 % level.
variances of Band C. The increment of variance in those two treatments,
therefore, should be ascribed to polygenic mutation induced by X-rays. An
increment in variance p8r unit dose was calculated by linear regression;
it amounts to 0.000167 for abdominals, but it is negative for sternopleurals,
although the variance in B is significantly larger than in A. A possible
explanation for the relatively lower variability between lines in C could besought in their lowered viability caused by the high dose of irradiation.
If mutations in the character analysed are impairing fitness, the line will
be eliminated during isogenisation. Evidences of lower hatchability andviability in the lines treated with a high dose support this possibility.
Taking the increment in variance due to spontaneous mutation to be
0.0047.5, which is only an average of the values reported by CLAYTON and
ROBERTSON (1955) and PAXMAN (1956), the tentatively calculated doublingdose in abdominal bristles was 56.89, or approximately 60 r.

43.

The Effects of X-ray Irradiation on Selection Response*(By Osamu KITAGAWA)

It has been shown in Drosophila melanogaster that the response to selection for a varying number of bristles was increased after X-ray irradiation(ScossmoLI, 1953; CLAYTON and ROBERTSON, 1955).
The purpose of this experiment was to determine the effect of X-rayson the acceleration of artifici2J selection. S~lection was conducted for highand low numbers of chaetae on the fourth and fifth abdominal plates offlies in an isogenic strain, onginating from the Oregon-R strain and designated the P line, and also in a hybrid between two isogenic strains,
namely Oregon-R and Samarkand, which was designated the Cline.
Selected parents were treated at each g~neration with X-rays of 1500 r justbefore mating. The high and low selected lines were classified in four

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

77

RESEARCHES CARRIED OUT E\ 1938

lots according to whether the treatment included: (1) both sexes, (2) only
females, (3) only males, or (4) neither sex. The selection intensity was
20% (6 out of 30 in each sex), except that no selection could be found insome later generations because of high sterility due to the irradiation.
After the twentieth generation, lots (2) and (4) of the P line showed
little or no response whereas some response (a few units in chaetae counts)
was obtained in lots (1) and (3). In the C line, a response was observedin all lots, an especially large response being obtained in lot (1). Lot (2)showed a response nearly equal to lot (3). These results seem to indicatethat the remarkable effects of X-rays on the induction of new mutationsin polygenic systems and on the increase of recombinations especially infemales might induce the new variations. The response in lot (2) of
the C line was strengthened by both, while in lot (3) the response was
induced mainly by the former effect. Furthermore, the mutation rate in
the polygenic system investigated under the influence of X-rays wasthought to be higher in the male than in the female.

44.

Breeding Structure of Poultry Flock which l\lIaximizes the Genetic
Progress by Selection(By Yukio YAMADA)

A typical poultry flock consists of offspring from a number of sires, eachmated to several dams. It is assumed that selection is from a populationof sdn female offspring originated from s sires each mated to d dams,
each mating producing n daughters.
Very recently OSBORNE (1957) derived selection indices for a sex-limited
trait, by which optimum gains may be obtained when individual, damfamily, and sire family averages are available for females but only dam
and sire family averages for males. Selection based on these indices istheoretically possible to achieve the largest progress compared with anyother type of selection, e.g. the combination selection of individual andfull-sib averages proposed by LERNER (1950).
Genetic gains per generation are generally expressed asAztlG= --h (fG

P

or

(1)

where zip is the intensity of selection and h or rax is the correlation of
genotypes and phenotypes. In the case of index selection rGX is thus
the correlation between the genotype and the selection index used. As
was already reported by the author (1958), the genetic gain can be partitioned into several parts according to the selection actually applied. If
different selection indices were applied for males and females, the expected
gain would be

RESEARCHES CARRIED OUT IN 195878'_ .dS+.dD.dG--f -1 r z,

Z2= 2 ·lp~RGll+ P2 RGI

}
2

(f/j

(2)

where ROIl and RCI 2 are multiple correlations of genotypes and indiceswhich were given by OSBORNE. Subscripts 1 and 2 refer to males andfemales, respectively.
Assume that the population size of females is N =sdn and the selection
intensities for males and females are fixed in every generation, and thatat least one male is retained for each dam family until they reach thebreeding age. Only one male for each selected family can, however, be
used as the breeder for the next generation so as to prevent inbreeding
progress, even though several males are available for selection in eachselected family. Thus the selection intensities are in turn

s
1
sd 1
P - sd -- d for males and P2=---=Nn for femalesl-and hence,

After substituting these into (2), we obtain another expression of .dG interms of p" pz, z" Zz, h/, N, and (fG. In order to get the maximum .dG we
differentiate it with P, and P2, and obtain a set of simultaneous equations. P, and P2 which satisfy the eqations, would be the optimum selection intensities for males and females of the breeding flock for a given
h2 and N. Consequently optimum numbers of families and daughters will
be determined. Details will be given elsewhere with numerical illustrations.

45.

Performance Test and Selection EjJiciency Involving
Different Environments
IBy Yukio YAMADA and Toshitaka ITO)

The present report deals with the performance tests of birds of similar
genotypes under different environments. Tests of this nature are import·
ant from the point of view of estimating the magnitude of testing station
effects (confounding with management) and also for the purpose of selecting birds when the family members are tested at several different places.
It is fairly common in this country to test the performance of birds forbreeding purposes under such conditions, mainly due to the small facilities
available to single breeders. In such cases, several breeders cooperatewith each other in testing and selecting birds on the basis of averagefamily records, little attention being paid to their individual performance.
However, if there were significant interactions between genotypes andRESEARCHES CARRIED OUT IN 1958

79

environments, the method used would not be appropriate for the purposeof selecting birds of superior genotypes under a specific environment.
The data were collected in 1957 from three breeding groups of Barred
Plymouth Rocks totalling 1580 birds. Chicks were hatched at the main
station, distributed to a number of branch stations within a small district
and reared under different management and housing. Hen housed egg
production of 500 days of age and the time of sexual maturity wereanalysed. Because of non-orthogonal data the usual method of analysis
of variance is not adequate to test the variances corresponding to specificeffects. Therefore," Method 1" described by HENDERSON (1953) was
employed.
The model used here was.
Y ijkl = IJ. +ii +sij+lik +(Sl)Uk +Cijk+Cijkl

where i, s, l, sl and C stand for breeding groups, sires, locations, interactions between sires and locations and errors, respectively. All factors otherthan the mean were assumed to be random variables. The sum ofsquares and coefficient of each variance component in these expectations
are presented in Table 2.
Table l.
GroupsSires

OffspringI

Statistics of the population analysed.
No. of testing I Sexual
locationsmaturity
involved!

Egg
productionEggweight196.55181.71

57.80
54.10
57.46

~-~--

14

21

B

9

771
276

11

199.68195.05

C
Total

9

533

13

200.65198.27

32

1,580

45

199.23

194.31

A~_

Table 2.
Source ofvariation

f.'

T-CT

56.96
-------~_.
_-

Sums of squares and coefficients of variance components.
2

2

I

(Je

i
!

Sexualmaturityi

Egg

I Production

, .!

'

.

975.76 1521. 57 11471.85 1566.72 1579 1568 735 00 I5862 689 73
975.76 101.53211.56
27.39
2
6,045.441 55,925.44
1420.0347.64 326.68
29
94,577.75 120,387.67
II

F-CT
S-F

L-F
SL-S-L+F
T-SL

..

1

85.17 1260.29
i

204.4742

275,611.50 i 303,500.18

1-85.17 -47.64 1018.18

258

158,422.231 886,994.93!

1248 1034,078.6814495,881.51

The solution to these equations is O"s2=52.66, 0"1 2= 194.21, O"SI2= -41.28
and O"e 2=828.59 for sexual maturity and O"s2=15.03, 0"1 2 =125.41, O"sz2=-34.90

80

RESEARCHES CARRIED OUT IN 1958

and 6 e"=3602.47 for egg production. These resulted in h"=0.204 for sexual
maturity and h"=0.016 for egg production. So far as relative magnitude
of variance components is concerned, the variances of interaction wereboth negative and thus the interaction would be unimportant, at least
under these conditions. Variances of station differences in both traits werefairly large but did not greatly affect the efficiency of selection. The error variance of egg production ,vas exceedingly large, this would be
ascribable to a variety of micro-environments within locations.
It is concluded that the method of testing birds under such conditions
is still valid but sources of environmental variation such as housing andmanagement should be unified or standardized as well as possible, to lessen
the environmental variation, and accordingly increase the efficiency of
selection.

46.

Evidence of Significant Interaction between Sire Family andDate of Hatching in Egg Production of Chickens(By Yukio YAMADA)

The cata utilized in the present report were obtained from the recordsof 15 sire groups of Barred Plymouth Rocks. Comparison of the influenceof different hatching dates was made on the basis of three wide periods
only; these extended from the 13th of February to the 10th of March,from the 11th of March to the 10th of April, and from the 11th of April
to the 29th of May. Parents used in the analysis were represented bytheir offspring in each period.
A preliminary analysis has shown that the variance of sub-classes was
highly significant. Further analyses of variance, using the method offitting constants to correct for disproportionality in subclasses, and themethod of weighted squares of means, were carried out. The results,
presented in Table 1, clearly indicate the significance of interaction between
sire family and the date of hatching for hen-housed egg production until
Table 1.

Analysis of variance for interaction and main effects by
the method of weighted squares of means.
d.f.

Hatches
Sires
Interaction
Error** Significant at 1 % level.

2

14
28
925

M.S.
25,606.21**
911.63
4,596.18**
2,670.69

81

RESEARCHES CARRIED OUT IN 1958

500 days of age. The same analysis was carried out in the case of sexual
maturity; however no significant interaction in this trait could be detected.
If the situation encountered here is a common one for prolonged hatching periods, the correction of egg production for the date of hatching, theestimation of heritability and improvement by selection, and the methodof multiple shift in poultry are not appropriate. Research on this kind ofinteraction is now under way and a discussion of its significance in poultry
breeding wiI! be given after the completion of the experiment.

47.

A Comparative Analysis of Body Weight in Two Purebreeds andTheir Reciprocal Crossbreeds in the Domestic Fowl
(By Takatada KAWAHARA and Shun ICHIKAWA)

During the three years from 1956 to 1958, intra- and inter-breed matings
involving White Leghorns (WL) and Barred Plymouth Rocks (BPR) werepracticed. Average coefficients of inbreeding were 3.4% and 9.2% respectively in WL and BPR. Data were collected from 762 purebreed and 586
crossbreed females originating from 16 sires and 141 dams in WL and 16
sires and 119 dams in BPR. Mating systems were such that these purebreeds and crossbreeds were half-sibs. Measurements of body weight weretaken at 0, 4, 8, 12 and 18 weeks of age. The mean body weights andtheir standard deviations for all surviving birds are presented in Table 1.
Table l.
-:-"----"'-'-----==:-c--=__

Mean body weights and their standard deviations.

------::-----=.::.-----=----------,-:-:------'--.:::"""-----...:..:==-'--"--------- --I

Breed or crossNo.
I startedI~---

Body weight (g)- ~ - -

0
week

8
weeks

12
weeks

560.1
95.39
625.7
112.30
616.4
77.15
620.0
81.94

861.9
146.00 i
1013.6242.90959.1
145.80995.0
143.00 !

18

I weeks~ - - _ . -

WL

435

BPR

309

WL¥xBPRej-

331

BPR¥xWL ej-

255

Total

1348

35.9
3.98
34.6
3.41
35.4
4.24
33.2
3.20

216.6
39.79
219.3
36.29
231.9
32.31
230.0
31.92

1174.7151.3
1466.0222.001302.1
164.201393.5120.50The significance of differences between mean body weights is given in
Table 2. A heterotic effect on body weight appeared definitely at the ageof 4 weeks; afterwards the body weight of crossbreeds approached that of

82

RESEARCHES CARRIED OUT IN 1958
Table 2.

Significance test of differences between means.

t values----.-------.

Comparison

o

week

WL vs. BPR

4:
8I
weeks I weeks!

8.40**
4.73** I 0.98
0.55
7.04** 0.70
1.69
5.69** 2.76**
9.42** 4.54** i 8.27**
4.58** 3.73**
I 2.66**
5.11** 3.62** 0.66

I

WUf·xBPR~ vs. BPRif.xWL~

WL vs. WLif. x BPR ~WL vs. BPRif. x WL~
BPR vs. WLif. x BPR~
BPR vs. BPRif. x WL~---:-:----::c:---:-::------,,-----:-__:_ --

---

12
weeks

18
weeks

10.10**
2.90**
11.00**

19.52**
7.14**
10.59**
18.57**
10.11**
4.40**

11.19**
3.37**
1.04

i.

-------** Significant at the 1 % level.

the mid-parents. The most interesting result is the finding ofdifferences in body weight between reciprocal F I hybrids at
/\
/
\
the
age of 12 and 18 weeks.
/
,,
/
/
,,
The "t" test shows that theI,,
J
BPRif. x WL~ cross gives signi,,
ficantly
heavier offspring than
....
:> ..
the reciprocal cross.
Q
Trends in variability of body10
weight in terms of coefficientsof variation (C.V.) are illustrated
in Fig. 1. During growth, thevariability of purebreeds is remarkably higher than in cross,2
breeds, especially in the period"
weeksfrom 4 to 12 weeks, which sugFig. 1. Coefficients of variation of body weight gests that a more effectivein pure breeds and their reciprocal hybrids homeostasis is operating on bodyat various stages.
growth in the F I hybrids, atleast from the 4th to 12th week of their development.
3'

a: WLif. x BPR t;b: WL
c: BPR
d: BPRif.xWL~/

48. Influence of Heterosis on Viability of F I Hybrids between
Two Breeds of the Domestic Fowl
(By Takatada KAWAHARA)A mortality analysis with data given in a previous report, excluding theT2 group (KAWAHARA 1957) and accidentally dead birds, was undertaken.

RESEARCHES CARRIED OUT IN 1958

83

The birds were maintained without any conscious culling up to eighteen
weeks of age. A summary of data with a significance test based on the
chi-square method is given in Table 1. The mortality of hybrid chicks
was 5.7% less than that of purebreeds, and this difference was significant
statistically at the 1% level. The mortality of BPR was 3.2% greaterthan that of WL (11.5% vs. 8.3%), and the mortality of F 1 hybrids ofa BPRif-xWL-& cross was 1.7% greater than that of the reciprocal (4.9%
vs. 3.2%). However, these differences in mortality were not significant
statistically.
Table 1.

Mortality of various mating types up to eighteen weeks of age.

Breed or crossNo.
Mortality
tested i -- No.

%

1.71

WL-

.'"

8.3

2.25

11.5

1

'"

7.14**
3.2
I

4.9

BPR

/
13.75** I

><

/

/

I

6.8k**'"'"'/'" x WL-&
WU;b !/
BPR-&-BPRif0.93

9.6
20.90**
3.9

E.
49.

MATHEMATICAL GENETICS

A iYlaximuJlz Principle in the Genetical Theory of Natural
Selection(By :Ylotoo KIMURA)FISHER'S "Fundamental theorem of natural selection" states that theTate of change of population fitness, measured in Malthusian parameters,
is equal to the additive genetic variance in fitness. However it does not

84

RESEARCHES CARRIED OUT IN 1958

specify how gene frequencies change in the process of natural selection
acting on genotypes.
The present report is an attempt to formulate a law of change in genefrequencies in the course of natural selection. Let AI, A" A!,···, An bea series of n alleles with respective frequencies of Xl, ;l", " ' , ;tn in a population. Under the assumption of random mating and constant fitness of
individual genotypes, it is possible to prove the following theorem: Fora given short time interval ot, natural selection causes gene frequencychanges 0~;1, OX2, "', OXn, in such a way that the increase of population
fitness shall be at maximum under the restriction±(OXi)" =}_ V (ot)'
Xi
2 (I
,

i-I

where V(I is the additive genetic variance in fitness.
The theorem can easily be extended to any number of loci. Furthermore, it may be extended to cover quite a general situation from which
the above restrictions of random mating and constant fitness of genotypes
are removed.
The most remarkable feature of this theorem is that the set of differential equations describing the law of change in gene frequencies bynatural selection can be derived from it. It may be called "a maximum
principle in the genetical theory of natural selection ", analogous to theprinciple of least action in physics.
(d. Kimura, M. 1958.

50.

Heredity 12: 145-167)

A Gene Frequency Cline Determined by Selection and Migration(By Motoo KIMURA)

Consider a population which is forming effectively a continuum ona linear habitat, where dominant individuals (AA and Aa) are advantageous
on one side and recessive individuals (aa) are advantageous on the otherside. If the range of migration of each individual per generation is verysmall as compared with the total range of distribution, a gene frequency
cline may be formed. For mathematical treatment, it may be convenient
to express a position on the habitat as a point on an one-dimensional
coordinate and take the neutral
position as origin. We assume
Dominants
Recessives
that
the dominants are advanta>Recessives>Dominants
geous on the negative or left sideof the coordinate axis while theXorecessives are advantageous on
the positive or right side (Fig. 1).
Fig. 1.

RESEARCHES CARRIED OUT IN 1958

85

Let u(x) be the frequency of the dominant gene A at the point x andassume that the selective advantage (or disadvantage) of the dominants
over the recessives at any point is proportional to the distance of that
Table 1.

x

Numerical solution of u" = xu(l- uj" with boundary conditions:
X=-= u=l,
x=+= 1£=0.

u

o

x

0.1
0.2
0.3
0.4

0.40115 i
0.37597
0.35103
0.326200.30191 '

1.0
1.1
1.2
1.3
1.4

0.5
0.6
0.7
0.8
0.9

0.278210.255250.233140.21200
0.19190

I

x

x

x

u

0.17294
0.155150.13858
0.123240.10913

2.0
2.1
2.2
2.3
2.4

0.048390.04172
0.035850.03072
0.02627

3.0
3.1
3.2
3.3
3.4

0.01009
0.00868
0.007540.006640.00595

1.5
1.6
1.7
1.8
1.9

0.096240.08452
0.07394
0.064430.05593

2.5
2.6
2.7
2.8
2.9

0.022410.01909
0.01625
0.01384
0.01180

3.50.00546
0.00517

x

u

x

u

0.96518
0.966480.96671
0.968890.97001---8.7 I
--8.8
-8.9
-9.0
- 9.1

0.98580
0.986110.986400.98668
0.98694

i

3.6

~r;

-0.1
-0.2
-0.3
-0.4

o

0.401150.42633
0.451290.476140.50052-2.9-3.0 I-3.1 I-3.2-3.3 i

0.87133
0.877900.884070.88987
0.89533

-5.8
-5.9
-6.0
-6.1
-6.2

-0.5
-0.6
-0.7
-0.8
-0.9

0.524410.54769
0.57033
0.59223
0.61334

-3.4
-3.5
-3.6
-3.7
-3.8

0.900470.90530
0.909840.914120.91815-6.3 I-6.4-6.5 I-6.6 I-6.7 I

0.971070.97208 Ir
0.97305
0.973970.97484

-9.2
-9.3
-9.4
-9.5
-9.6

0.987200.987450.987700.98794
0.98818

-1.0
-1.1
-1.2
-1.3
-1.4

0.633620.65306
0.671630.68934
0.70618

-3.9
-4.0
-4.1
-4.2
-4.3

0.921960.92556
0.92894
0.932140.93517 !

-6'8 I
-6.9
-7.0
-7.1
-7.2

0.97568 . -9.7
0.97647 i -9.8
0.97723 i -9.9
0.97795 -10.0
0.97864 -10.1

0.988400.98861
0.988810.98900
0.98918

-1.5
-1.6
-1.7
-1.8
-1.9 I

0.722170.73732
0.75166
0.765210.77800

-4.4
-4.5
-4.6
-4.7
-4.8

0.938030.94073
0.94329
0.945700.94798-7.3-7.4 i-7.5 i-7.6-7.7 I

0.97930
0.97992
0.98052
0.98109
0.98163

-10.2
-10.3
-10.4j-10.5 I
-10.6 .

0.989540.989500.989640.98977
0.98989

-2.0
-2.1
-2.2
--2.3
-2.4

0.79006
0.80143 i
0.812130.822200.83169

-4.9
-5.0
-5.1
-5.2
-5.3

0.950140.952190.954120.95595
0.95769

-7.8
-7.9
-8.0
-8.1 i-8.2

0.982140.98263
0.983100.98355
0.98397-10.7-10.8 '
-10.9
-11.0
-11.1

0.99000
0.990090.990180.99026
0.99033

-2.5
-2.6
-2.7
-2.8

0.84062
0.84902 .
0.85692
0.86435

-5.4
-5.5
-5.6
-5.7

0.95934
0.960900.96239
0.96381

-0.3
-0.4
-0.5
-0.6

0.98438

I

I

j

I

O.98~76

0.985120.98547 Ii

I

-11.2
0.99038- 11. 30 .99044- 11. 4 i O. 99050
I

86

RESEARCHES CARRIED OUT IN 1958

point from the ongm. Then it is possible to show that, by choosing
a proper unit of length to measure x, u(x) at equilibrium satisfies the following second order differential equationd"u =XU(l-l!I"dx"'

with boundary conditions
x=-=

u=l

x=+=
u=O.
Because of the non-linearity of the above equation, it would seem to bevery difficult to obtain an analytical solution. So numerical analysis was
employed to get values of u(x) correct to five decimal places, from :t = -11.4,
by intervals of 0.1, to x=3.6. The values are tabulated in Table 1 and are alsoplotted on a solid line in Fig. 2. lowe the numerical analysis to Dr. Shozou.(P)=--------10.0 -9.0

-8.0

-7.0

-6.0

-so -4,0

-3.0

-2.0

-1.0

0

1.0

2.0

.30

4.0;(

Fig. 2. A graph showing the gene frequency cline (solid line) and the phenotype frequency cline (dotted line) determined by selection and migration whena pair of alleles with complete dominance are involved.
SHIMADA operating the HIP AC 1 (Hitachi Parametron Automatic Computer1) at the Hitachi Central Research Laboratory, to whom I wish to express
my deep appreciation for his help. The value of u(x) and its slope u'(x;at the origin are as follows:u(01=0.401152
u' (0) = -0.252046 •The frequency of the dominant individuals may be calculated by
P=2u-u" ,values of which are plotted on a dotted line in Fig. 2.

RESEARCHES CARRIED OUT IN 1958

51.

87

Conflict between Set/-fertilization and Outbreeding in Ptants(By Motoo KIMURA)

It has long been known, and was even noted by DARWIN, that seedlings
produced by crossing two different individuals of the same species areusually more vigorous than the ones produced by self-fertilization.
On the other hand, there are certain circumstances in which self-fertilization is favored by natural selection. For example, where the density of
individuals is very low, self-fertilizing devices such as cleistogamy arecertainly of advantage as they ensure seed-production. Also, the formation of amphidiploids more vigorous than their parental species is known
to be one of the important means of species formation with which plants
are specially endowed and since the capacity of self-fertilization is essential
for the establishment of new amphidiploid species, such ability will bepromoted or at least preserved in the course of evolution.
A more interesting factor promoting self-fertilization may be the occurrence of genes which enable a plant to be fertilized by its own pollen
without losing its power of fertilizing other plants. Let Al and A z bea pair of alleles with respective frequencies of x and I-x in the population, and let Pu, ZP12 and P n be the respective frequencies of AlAI, AlA z
and A 2A 2. We assume that in AlAI individuals, ovules are fertilized bypollen grains randomly taken from the entire population, while in A z A 2
individuals they are self-fertilized with probability S and out-crossed withprobability I-S. In the heterozygous individuals (AlAz), the probabilityof self-fertilization is assumed to be Sh where h represents the degree ofdominance of A z gene. If D is the amount of inbreeding depression of
the seeds produced by self-fertilization such that I-D is the relative
viability of the selfed seeds as compared with the seeds produced by theout-cross, then it can be shown that the rate of change of the frequencyof Al per generation may be given by
JX=S(D-t ){ZhP12(

x-t)+P22X} ,

W

where

w=I-SD(ZhPlz+P2z)
is the average selective value of the population. If Jz lies between 0 and
1, the term in the braces of the above equation is non-negative and since
Sand ware both positive, the right side of the equation' is positive or
negative according as
D-l/Z

88

RESEARCHES CARRIED OUT IN 1958

is positive or negative.

Thus, ifD<1/2,
Ltx>O

unless x(l-x)=O and an intermediate gene frequency will lead to x=l.
Namely the self-fertilizing gene A 2 will eventually be lost from the population. On the other hand, if
0~D<1/2 ,Ltx is negative and the gene A 2 will be fixed in the population. If D=lj:?',however, an intermediate gene frequency will be held at neutral equilibrium.
It is interesting that these conclusions depend only on the value of D
and not at all on Sand h. Since on the average half of the heterozygous
loci become homozygous after one generation of selfing, the condition
D=1/2

will be realized if the population contains recessive deleterious genes
amounting to one lethal equivalent per gamete. Thus we may say thatan outbreeding plant species is immune to the attack of self-fertilizing
genes if it contains recessive deleterious genes in the amount of more than
one lethal equivalent per gamete or two per zygote.

52.

Inverse Approach to the Estimation of Genetic Load
Discolosed by Inbreeding(By Motoo KIMURA)

The method which I propose in this report for the estimation of thegenetic load through a consanguinity study may be called the inverse or
retrospective approach, in the sense that the distribution of various typesof inbreeding is investigated retrospectively after a sample of affectedindividuals has been collected. On the other hand, in the usual approach,which may be called the direct or forward approach, incidences of abnormalities or mortality are investigated among individuals produced fromdifferent degrees of consanguinity. It is this forward approach which wasused recently by MORTON, CROW and MULLER to assess the genetic load interms of lethal equivalents. They estimated parameters involved in the
regression of mortality on the coefficient of inbreeding. Their contribution
is outstanding, especially in bringing to light the genetic implication ofsuch parameters.
The inverse or retrospective approach is based on the rather obvious
consideration that consanguinity would be higher among affected than un-

RESEARCHES CARRIED OUT IN 195889

affected individuals, and by studying the amount of difference one could
asses the hidden genetic load involved.
In what follows, I will explain, using neonatal death as an example,
how the parameters relating to the genetic load may be estimated by thisapproach, using the method of maximum likelihood.
We assume that, out of the total (T) births registered, M cases ofnatural neonatal death are reported, and 1''11' of them are available for the
study of consanguinity, among which m/ cases are found to be the resultsof marriages between individuals of relationship Ii (i=O, 1, 2, ... ).

MI=mo/+ml +m{ + ....
lSuppose also that we extract a random sample of size N from T births
to investigate the distribution of the inbreeding coefficients and find thatni (i=O, 1, 2, ... ) of them have the inbreeding coefficients Ii, 'iNhere

10=0,/I=1/16,etc.

Let Pi be the rate of neonatal deaths among babies with the inbreeding
coefficient Ii such that

Pi=A+Bli;then the joint likelihood of getting the above samples is
L=Const. x{ n (CPi)mi'}pll-31'\l-PY-M{ n Gin i} ,
i=O

i=O

where P is the rate of neonatal death in the general population with theinbreeding coefficient f, i.e.

P=A+B!,and Ci is the proportion of marriages with the relationship Ii, such thatf= ~ Cdi.
i= U

After eliminating the unnecessary parameters of Ci'S, we obtain thefollowing set of equations to give the maximum likelihood estimates of A
and B.

I

The above equations may be solved by successive approximation.
'be convenient to use/
A=MNmo and B=16NM(M' no -Nmo )'Af TnoTn oll/I'(N-no)

It may90

RESEARCHES CARRIED OUT IN 1958

as the starting values, which may be obtained by neglecting consanguineous
marriages other than first cousin marriages.
The error variances dA 2 and dB 2 and the error covariance dAB of theabove estimates may be obtained by the following equation:
2FAF B
FAGB+FBGA
2GAGB

where
F A--"!l'L
L...l p2 ''-0!

in whichIm{lti = M'

F.

53.

,

p=M
T

and

BIOCHEMISTRY OF PIGMENTS AND OTHER
SUBSTANCES IN PLANTS

Biochemical Genetics of Flower Color in Swiss Giant Pansies,
Viola X Wittrockiana Gams
(By Toru ENDO)

1. Chemistry of anthocyanins
In the common garden varieties of Swiss Giant Pansies, flower colorsranging from red to blue are caused by at least six anthocyanins which.

RESEARCHES CARRIED OUT IN 195891

are arbitrarily designated as aCl, aD 2 , C3 , D., C5 and Do. They wereseparated from each other by mass paperchromatography in a comparatively
pure state. Their aglycone, sugar and organic acid components, and sugar
residues were studied chromatographically after complete as well as partialhydrolysis, and also after saponification with alkali. The results are summarized as follows:aC l : cyanidin-? -p-coumarylglycoside,aD 2 : delphinidin-3: 5-p-coumarylglucorhamnoside,
C3 : cyanidin-3-g1ucorhamnoside (keracyanin),
D4 : delphinidin-3-g1ucorhamnoside (tulipanin),
C5 : cyanidin-3: 5-g1ucoglucorhamnoside,
Do: delphindin-? -glucorhamnoside.
Among these anthocyanins, the major pigment in reddish varieties is
Cs and the pigment in bluish varieties is aD 2 which is not violanin(dephinidin-3-p-coumarylglucorhamnoside) but a new anthocyanin, as mentioned above. The others are minor pigments. Anthocyanins in blotchedparts of the flower petals are chiefly aD 2 together with a small amount ofaCl (Bot. Mag. 72: 10-19).
2. Dominance relations in F 1 hybrids, with special reference to flower
color and anthocyanin pigment constituentsSeven varieties with different flower colors were crossed and approximate dominance relations were observed as follows:

t:::\/~'-f---+---",---_.

\:;)~ . .

j\

RESEARCHES CARRIED OUT IN 1958

92

a major anthocyanin, aD 2 , is dominant over that of another major anthocyanin, C3 : (3) some anthocyanins are quantitatively increased, decreased
or inhibited in F 1 hybrids among the cyanic varieties: (4) on the chromatograms, everyone of the anthocyanins found in the F 1 hybrids corresponds to one present in the parental varieties. (Jap. J. Genet. 34: 116-124).

54.

Qualitative and Quantitative Analyses of Bitter Substancesin the Ripening fruit of Citrullus colocynthis(By Yoshito OGAWA and Kazuo FURUSATO)

The results of qualitative and quantitative analyses of bitter substances
found in the ripening fruits of Citrullus colocynthis are reported.
The flesh of the fruits was mashed and treated with methanol 5, 10, 15,
20 and 30 days after flowering. After the evaporation of the solvent,remaining dark green oil was treated with chloroform by which the bitter
substances were completely absorbed. Then they were subjected to paperchromatography by the ascending method with water-saturated ethylacetate at 20°C. All of the materials were fractionated into 41 parts. All
factions were dissolved in water and their taste threshold was examinedby the same method as used in the study on Swertia chirata by KORTE(1955).
In ripe fruits, only one bitter substance was detected. This substance
was identical with" Citbittol ", formerly isolated by the writer (1957). In
Table 1.

Variation in the amount of bitter substances in the ripening
fruits of Citrull718 colocynthis, Schrad.
(Bitter value)
Citbittol

Days after
floweringA

0.90*
191.6 (17.2**)
59.0 ( 9.4**)
27.8 (10.3**)
14.6 (10.9**)

5
10
15
20
30***

,

1.0 ( 1.0**)

1 B
I

C'

D

E

F

G

I

H 1

0.70* 0.61*1 0 . 54* 0.44*;0.38* 0.30*1'0.15* Total
I'0.61 -;;-

2.4

3.0

2.4 I 0.·0

2.4

3.6

0.4!

0.2
0.0
0.0

1.2
0.4
0.0

0.4 1 0.0 1 0.0 I 0.0
0.0
0.0 I 0.0
0.0
0.0
0.0
0.0
0.0

0.6 1 0.2 1 0.2

I

0.0 200.2
0.2 ! 66.6
0.0 29.6
0.0 15.0
0.0
1.0,---------_.

* R f of paper-chromatography with water-saturated ethyl-acetate byascendence
method at 20°C.
** Amount of Citbittol A per fruit.
*** Ripened.

93

RESEARCHES CARRIED OUT IN 1958

unripe fruits, seven new bitter substances were found in addition. The
former name" Citbittol" was then changed to "Citbittol A", and the
newly found bitter substances were called" Citbittol B, C, D, E, F, G and
H". Citbittol A, however, formed over 90% of the total of bitter components.

55.

Bitter Substances in the fruits of F I Hybrids between
Watermelon and Colocynthe(By Yoshito OGAWA and Minoru SHIMOTUMA**)

Research on the bitter substances contained in the fruits of the F I hybrids
between Citrullus vulgaris Schrad (2x, 4x) and Citrullus colocynthis Schrad(2x) was carried out by the same method as was used in the experimentwith Citrullus colocynthis. ***The methanol extract of the fruits of the hybrids was analysed bypaper-chromatography with water saturated ethyl-acetate and the bittervalue was calculated.
Table 1.

Bitter substances in the ripe fruits of F I hybrids between
watermelon and colocynthe.
(Bitter value)
Citbittol

Cross combination

D-I-E-'-~-IBic

1- - -: 0.90* iO.70*10.61* 0.54*)0.44*1 Total---------------•

C. vul. (Otome, 2x)x C. colo. (No.1, 2x)
C. vul. (Fumin, 2x)x C. colo. (No.1, 2x)
C. 1ml. (Yamato-cream, No.2, 2x)x C. colo. (No.1, 2x)
C. vul. (Ashi-yamato, 4x)x C. colo. (No.1, 2x)
C. vul. (Yamato, 4x)x C. colo. (No.1, 2x)
C. vul. (Fuken, 4x)xC. colo. (No.1, 2x)~~~.~~~~-:~.-;-! -7~33.0

0.0!
0.0 1 0.0

0.0

33.0

106.8

3.8

0.6 I 0.0

0.0

111.2

1.8

I
0.6 I 0.6

0.2

88.2

0.0 I 0.0 ['

0.0 I

43.8

0.2

58.6

0.0

82.0

C. colo. (No.1, 2x) (Standard)
0.0 I
0.0
0.0'
______________________________.. __L ..

1.0

C. colo. (No.1, 2x)
xC. vul. (Asahi-yamato, 2x)

I

85.0
42.0

1.8

I

I

I

56.4 1 0.6 \ 0.4 I

1.0
1

81.0

0.2

_

* R f of paper-chromatography with water-saturated ethyl-acetate by ascending
method at 20°C.
*****

Kihara Institute for Biological Research, Yokohama.
Refer to Report No. 54.

RESEARCHES CARRIED OUT IN 1958

94

The concentration of "Citbittol A" in the ripe fruits of the hybrids isgenerally higher than in Colocynthe. The bitter substances of the hybrids
are different and their concentrations vary according to the variety ofwatermelon examined as shown in Table 1.
The watermelon has no bitter substance. The bitter substances detectedin the fruits of the hybrids have been already identified in the Colocynthe
parent. *56.

Anti-cancer Activity of "Citbittol A "(By Yoshito OGAWA)

LAVIE et a!. (1957) reported that" Elatericin B ", a glycoside isolated from
Cucurbitaceae (Ecballium elaterium and Citrullus colocynthis), showed at 4
gamma per 1 g. body weight a good tumor controlling effect on Sarcoma
37 transplanted into mice. "Elatericin B" (kindly supplied by Dr. LAVIE)
is very bitter to the taste.
In the present study, the anti-cancer activity of another Cucurbitaceae
component was tested against an ascites tumor, Yoshida sarcoma, transplanted into a strain of rat, Wistar (two months old). The Cucurbitaceae
component tested was "Citbittol A", a bitter substance isolated by thewriter (1957) from the fruit of Citrullus colocynthis Schrad.
Immediately after the transplantation of the sarcoma, Citbittol A (50gamma or 5 gamma per 1 g. body weight of rat) was injected at the axilla
sub::utaneously, and the effect of this substance on the sarcoma wasobserved, especially in the following three points:
1) Mitotic activity in sarcoma cells,2) Systemic symptoms of the rat bearing the sarcoma (appetite, ascites
icterus and metastasis),3) Prolongation of the host's life.
In the mitotic activity of sarcoma cells, a transient decrease (significant
at 5% level) was observed 48 hours after injection. As to the systemic
symptoms of the host rat, no difference was recognized between the treated
and the control groups in the first seven days. But 10 days after injection,
the treated group showed a remarkable recovery and the animals remained alive for more than 100 days after transplantation of the sarcoma
cells, except for one rat which died 17 days after injection. The control
group was extinct on the 10th to 13th day. No toxicity of "Citbittol A"
in the above mentioned doses was observed either at the injected site orin systemic symptoms.

* Refer to Report No. 54.

.

RESEARCHES CARRIED OUT IN 1958

95

This experimental result may reaffirm the anti-cancer activity of Cucurbitaceae components. Biochemical and pharmacological comparisons between "Citbittol A" and "Elatericin B" are now being made.

G.

GENETICS AND BIOCHEMISTRY OF MICROORGANISMS
57. Curly Flagellar Mutants in Salmonella(By Tetsuo IINO)•

A mutant strain, SW577, of S. typhimurium R (FREIWER & LEIFSON, 1952)
produces curly flagella in phase-I, i-phase bacteria, and normal flagella inphase-2, 1.2-phase organisms. The cells with curly flagella rotate in liquid
media and aggregate. On semi-solid media, however, they cannot swarm.
In contrast with paralyzed mutant cells, they are sensitive to chi-phage.
The strain reverts rarely and then produces cells with normal flagella inboth phase-l and phase-2. The antigen types in each phase are identicalwith those of SW577.
Transduction was carried out from the phase-2 culture of the diphasicstrains to the phase-l culture of SW577 mediated by phage P22. Phase-lcells with normal flagella were selected on semi-solid medium containing
anti-1.2 serum. The normal type transductional clones obtained manifest
the antigen type of the donor in phase-l and that of the recipient in phase2. That is, the gene which controls the curl of flagella in phase-l is notseparable from H 1 by recombination. In transduction from SW577 to thestrains with Ah1 - (SWI061, SW629 and SW547; see the Annual Report,
1957), recombinants between Ah1- and the curl controller were obtained.
A parallel type of mutant in phase-2 was isolated from S. abortus-equi
SL23. The mutant strain, SJ30, produces curly flagella in phase-2. The
curly character is transduced linked to H 2 •From these results, together with the results obtained previously (theAnnual Report, 1957), it is inferred that H 1 and H 2 are the primary templates of flagellar protein (flagellin) in phase-l and phase-2 respectively;
mutations in H, or in H 2 produce the altered configuration of flagellin,
resulting in a change in antigen type, modification of the flagellar morphology or the inhibition of flagellar morphogenesis.

96

RESEARCHES CARRIED OUT IN 1958

58.

Cistr01l Test of Motility Genes in Salmonella(By Tetsuo IINO)

The trail phenomenon in the transduction of motility (STOCKER, 1956;
LEDERBERG, 1956) was applied as a test of the cis-trans position effect between the motility genes of Salmonella. In sixteen non-motile mutants
which were obtained from S. typhimurium TM2, the production of swarms
and trails in reciprocal transductions was examined on semi-solid media.
The strains, SL499, SL478, SW1153 and SJ60, are paralyzed types; thatis, they are flagellated but non-motile. Transductions between them produce swarms but no trails at all, indicating that they are mutants ina cistron. The cistron will be given the symbol l'fOt. The remaining 12
strains are non-motile and non-flagellated. Two of them (SL482 and SJ35)
are mutants in the Fla, cistron, which is linked to Hi. Each of the four
strains, SL480, SL481, SW1154 and SJ31, produced both swarms and trails
in transductions to and from any other strains tested. The results ofreciprocal transductions among the remaining six mutants are summarized inTable 1. Under the hypothesis that the mutant genes in these six strains
Table 1.
Recipient
donor
SJ33
SJ34
SL488
SL490
SJ36
SL483
TM2 (Fla+)

Reciprocal transductions between six non-motile mutantsof S. typhimum;um TM2.
SL490

SJ36

SL488

o

1

2

1

1

2

3
3

o
o

o
o

1

2

1

1

2

2

2

1

o

2

3

2

3

2
3

o

3

3
3

1
2
3

o

o

o

o

o

o

SJ33

SJ34

SL488

o

1

1

o

o

1

1

0: neither swarm nor trail. 1: swarms.
short trails. 3: swarms and trails.

3

2: swarms and a small number ofare complementary but the complementarity declines as the distance between the t\yO mutant sites increases, the sequence of the mutant sites isinferred to be as follows: SJ34-SL488-SJ36-SL483; SL488 is a mutant which
includes the sites of both SL33 and SL490.

RESEARCHES CARRIED OUT IN 1958

59.

97

Pteridine l'vletabolism in Aspergillus oryzae(By Saburo NAWA)

A purple fluorescent substance produced by a strain of Aspergillus
oryzae was characterized as a derivative of 2-amino-4,7-dihydroxypteridine(I) from its ultraviolet absorption spectrum and chemical nature. When
A. oryzae was grown on Czapek-Dox's medium supplemented with extra
2-amino-4-hydroxypteridine (II), two fluorescent compounds were isolated
as metabolites of (II). One of these compounds, which is bright blue
fluorescent in alkaline solution, had an ultraviolet absorption spectrum very
similar to that of 2,4-dihydroxypteridine (III). The second purple fluorescent substance was identified as 2,4, 7-trihydroxypteridine (IV), paperchromatographically and spectrophotometrically. It was observed that the rapid
deamination of (II) to (III) occurs in homogenates of the mycelium. Enzymes, partially purified by precipitation with acetone and dialysis, were also
capable of catalyzing the reaction. Furthermore, (III) was oxidized to (IV)at very slow rate in both homogenates and enzymes. Therefore, it seems
that in vivo (II) is first deaminated to (III) by pterine deaminase and ultimately oxidized to (IV) by pterine oxidase.
It was also found that the pterine oxidase is capable of oxidizing (II) to(1) and that (I) can not be acted upon by the deaminase. Therefore, theevidence that no trace of (I) could be detected in metabolites of (II) suggests that the activity of the deaminase is much higher than that of
the oxidase. Derivatives of (II) substituted in the 6-position were also
converted to substituents of (III) by the deaminase.
From these observations, there are the two following possibilities; i) A
naturally occurring pteridine in A. oryzae carries some substituents which
prevent attack by the deaminase, assuming that the pteridine is derived
through an oxidation pathway of the (II) family. ii) Otherwise, thepteridine is synthesized by different pathways from oxidation of the (II)family, in case of the absence of such substituents.

98

RESEARCHES CARRIED OUT IN 1958

H.

IV.

RADIATION GENETICS IN ANIMALS

60. Radiation Mutagenesis in the Silkworm
Independence of Radiation Intensity as Shown by Induced
Sterility of Spermatogenic Cells*(By Yataro TAZIMA)

Silkworm spermatogenic cells show a hyper-sensitivity to radiation, as
revealed in terms of excessive sterility, on about the second day of thefifth larval stadium, a few days earlier than the peak stage of radiationinduced mutability. The existence of separate sensitive periods for thetwo types of radiation response suggests that the cause of excessive sterility is different from that of induced mutation.
Moreover, the hatchability curve for pigmented eggs, plotted against stageof irradiation, remains almost constant at the same dose of X-ray irradiation with the exception of the excessively sterile stage. This result showsthat the factor causing excessive sterility must be different from the factor inducing dominant lethals which are presumably produced by grossstructural changes in the chromosomes.
Therefore, physiological damage of the cell as a whole was assumed asa possible explanation of the hyper-sensitivity of irradiated cells at thisparticular stage (T AZIMA, 1958).
According to this hypothesis, radiation would affect germ-cells throughmultiple hits at this stage, eventually killing them. The validity of thisassumption was tested by irradiation experiments with different intensities.
An experiment was carried out with 0.3 r/min r-rays and 62 r/min X-rays,
keeping the total dose delivered the same (800 r).
The r-rays from 60CO were delivered to the germ-cells of fifth instar
larvae from the ages of 11 to 56.5 hours for 45 hours, except for several
short breaks for feeding. X-ray irradiation was carried out at one of thethree checking stages, i.e. at the ages of 11, 33.6 and 56.5 hours. The
duration of the X-ray treatment was 12.9 minutes.
Two of the X-rayed groups were affected less than the one irradiatedat 33.6 hours, whereas in this group the effect of acute irradiation was
found to be of about the same degree as that of the r-irradiated group,
even though the radiation intensity differed by a ratio of 207: 1.
These results show that the effect of radiation on sterility of germ-cells
is independent of intensity, and is cumulative with the doses delivered.
This result is in contradiction to the expectation from the multiple hit* This work was supported by Grant RF 57178 from the Rockefeller Foundation.

RESEARCHES CARRIED OUT IN 1958

99

hypothesis. The above working hypothes is being disproved, it could besuggested that radiation-induced disturbance in the synthesis of basic cellular constituents, such as DNA, may be the cause of the excessivesterility of the germ-cells.

Radiation Mutagenesis tn the Silkworm
Further Data on Radiation-induced Mutation Rates
in the Silkworm*
6] .

V.

(By Yataro TAZIMA and Kimiharu ONIMARU)

It was reported previously (T AZIMA, 1958) that visible mutation rates at+pe and +"e loci of the silkworm 'vvere markedly higher than the mutation rates found in Drosophila. The mean mutation rates obtained at theseloci were about 7 times as high in the mature s'perm and 28 times as highin spermatogonium as the rates reported by ALEXANDER (1954) for Droso-

phila.
In calculating mutation rates in Drosophila, ALEXANDER excluded all
phenocopies, partial expressions, sterile variants and dead before completion of the test, while in the silkworm most of these categories were notexcluded from the calculation. It must also be noted that in the silkworm
the mutation rates were obtained for mutant characters expressed in theegg stage, in contrast to those obtained for adults in Drosophila.
Survival of egg color mutants was, therefore, examined in comparison
with normals in the same lots. The survival indices of pe and re mutantsfrom females irradiated with 2000 r were 0.78 and 0.56 respectively, giving
0.67 as an average. Even if we take 0.5 as the most conservative estimate of survival rate and 0.5 as the fraction covering phenocopies andothers, which are not transmitted to the offspring, we still get higher
mutation rates for the silkworm than for Drosophila.
Hence, another experiment was carried out using chocolate, a body color
mutant for newly hatched larvae, as a marker. The results are shown in
Fig. 1 and Table 1.
The mutation rates according to stage are in this experiment more orless similar to those obtained in the previous experiment with egg color
mutants, even though they are lower.
The rates obtained are still 3.5 times as high in spermatogonium and6.7 times as high in mature sperms as those in Drosophila. The survivalindex for the detected mutants was higher than for pe and re, and approximated 0.80.

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

RESEARCHES CARRIED OUT IN 1958

100

II>

::l
C)

~ x10-7... 15Control
o 2.47 10- 5~ 0.00

<ll

<ll

'"...

~

...

n:l

<ll

... '" 10

lV-2

111-3

V-2 V-4v-o v-a

PE

PM

PL

Irradiated stage

Fig. 1. Changes in radiation induced visible recessive mutation rate at
locus with the development.
Table 1.

Spontaneous and radiation-induced mutation rates per r at +Ch locus.
I

Sex

+ck

I

I

Spont. mutation
rates:~:le-·-I-~:~~xlO-5

Radiation-induced mutation rates
Gonium0.42 X 100.53

7

i Matured egg or sperm
17.5 x 10- 7

4.0

However, it is not as yet conclusively determined whether or not mutation rates are higher in the silkworm than in Drosophila.

RESEARCHES CARRIED OUT IN 1958

62.

101

Histological Study oj Radiation Sensitivity of Spermatogenic
Cells of the Silkworm (a Preliminary Note)*(By Toshihiko SADO)

In a recent report by T AZIMA (1957) it was demonstrated that theincidence of radiation-induced sterility in the silkworm differs with sex
and age of the irradiated insect, and that an extremely sensitive stage
occurs early in the fifth stadium of the male. However, no cytologicalstudy of the cause of these phenomena had been made. Therefore, thepresent experiment was undertaken.
Various doses of X-rays (100 r, 500 r, 1000 rand 4000 r) were given to
silkworm larvae at the early fifth stadium with a dose rate of 147.7r/min(180 kV, 25 rnA, 1.0 mm AI-filter, 50 em distance), but some younger stages
also were subjected to X-rays. Testes from irradiated animals were fixedat definite intervals after irradiation, and examined following the usual
histological methods.
Several hours after irradiation pycnotic or necrotic cells were observedin the gonial region, increasing in number
from the time of irradiation up to 24
hours, when the highest incidence wasobserved (Fig. 1). The incidence of thesedegenerating cells was raised with increasing doses. Later, the necrotic cells
rapidly liquefied and reduced the numberof spermatogonia in the gonial region of
the irradiated testes.
In a testis weakly irradiated at 100 r,
recovery was observed in the depleted
gonial region, while in testes heavily irradiated at 4000 r the ability to recover
was reduced to such a great extent thatscarcely any spermatogonia were observed
around the apical cells, even 11 days
after
irradiation. In moderately irradiatedFig. 1. Necrotic cells observed inspermatogonial region 24 hours groups, regeneration of spermatogonia
after X-irradiation at 1000 r on was found seven days after irradiation.
the 2nd day of the 5th instar.
The most sensitive of the irradiatedMost of the late spermatogonia
spermatogenic
cells were late spermatoare necrotic.
gonial stages.

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

102

RESEARCHES CARRIED OUT IN 1958

Spermatocytes were less sensitive to X-rays than were spermatogonia,
throughout the experiment. Cells irradiated at the former stage underwent apparently normal meiotic division without showing any necroticchanges, but as they turned into spermatozoa they showed nuclear abnormalities.
The apical cells located at the blind end of each testicular follicle were
found to be resistant to radiation.
The reduced fertility of irradiated animals appears to be partly due toa lack of sperms as a result of spermatogonial depletion and partly tothe formation of abnormal sperms which were not able to perform
fertilization.

63.

Studies on Mutation Rates after Chronic Irradiation 111 Mice.
A Preliminary Report on Sex Ratio.
(By T. SUGAHARA, K. TUTIKAWA and T. TANAKA)

Male mice of a multiple recessive stock were irradiated with gammarays for three successive generations as proposed by]. B. S. Haldane,in order to obtain animals which would accumulate recessive mutations.
The adult male mice were reared in the irradiating room, exposed to
gamma-rays from 'iDCO at a dose of 7.8 to 5 r, 22 hours per day for 30 to
45 days. The mating system and irradiating conditions are shown in
Fig. 1. Irradiated animals are marked with asterisks. G denotes thedominant wild stock and g the multiple recessive one.

p.
~

P2
PI
1",

gg'i!- xggti*
gg'i!- xgg*
x
gg'i!gg1;*
gg'i!(;-e}x
Gg x Gg

Total dosein r
167
gg'i!- xgW~*
gg'i!- xgg1;*x
gg'i!1~
001;*
x
225
gg1;*

rm

Periodin days

30
32
45

0
0

In this report, the sex ratio in each generation was studied for three
to six months after the end of irradiation. The multiple recessive stock
mice belonged to the NH strain, and those of the wild type were CBA.
The number of litters obtained, mean litter size, and sex ratio in eachgeneration are shown in Table 1. The increase in the proportion ofmales in the first generation and its gradual decrease in the succeeding
generations are considerable, although the statistical errors are rather too* This work was supported partly by Grant RF 57178 from the Rockefeller Foundation.

RESEARCHES CARRIED OUT IN 1958

103

large to make the data statistically significant because of the small
number of animals used. Variations in the mean litter size were notalways parallel with changes in the sex ratio. The reason for the discrepancy is not clear.
Table l.
Control

P4

P3

P2

No. of litters
47
No. of progeny
253
Mean litter size
5.4
Sex ratio
0.538
Probable error ±0.031

22
88
4.0
0.570
±0.054

41
188
4.6
0.547
±0.036

45
189
4.2
0.505
±0.038

Pl
~

'(1)'

¥

17
77
4.5
0.506
±0.056

29
187
6.4
0.427
±0.036The deviations of the sex ratio may be interpreted as follows, on theassumption of sex-linked dominant and recessive lethals induced byirradiation. The mutation rate for sex-linked dominant lethals is denotedby IIIfr, and that for sex-linked recessive lethals by 1-'-2/r, according to
the hypothesis that a linear relation exists between dosage and mutations.
Radiation doses for each generation are denoted by D 1 , D 2 , and D 3 • The
deviations of the sex ratio from that of the controls in P4, P 3 , and P 2
were Lh, .d 2 , and .d3 respectively, while in Pr the difference in sex ratio
between gg males and gg females was .d 4 • Then, we have as a good
approximation;,u 1 D 1 =4.d 1
1,u 1 D 2 - "2 D 1 ,u2 = 4.d2

,ulDs-( ~ D 1 + ~D2),u2=4.d3-(i

D 1 + ~ D 2 + ~ D3),u2 = 4.d 4

From the above equations, using the method of least squares for our
experimental results, we have
,ul=7.1xlO- 4,u2=2.0xIO- o

The .d s observed and those calculated with these values for ,ul and ,u2 arein good accord with each other in the range of the statistical errors asshown in Table 2. Further study of the sex ratio is now under way inorder to examine the correctness of our assumption.

104

RESEARCHES CARRIED OUT IN 1958
Table 2.

Deviation of sex ratioPI

Observed
Calculated

64.

Pi

P:l

P2

.032
.030

.009
- .009- .03:3- .018

~

'(!)

-9-

/
/

- .079- .081

Effect of Radiation on Living Cells in Tissue Cultures(By Masakatsu HORIKAWA* and Tsutomu SUGAHARA)

It is well known that the primary effect of radiation on Drosophila
larvae is not killing of the irradiated larvae, but a delay of pupation
and a decrease of pupation and emergence percentages over a wide rangeof radiation doses.
Furthermore, it has been observed by the authors that the body weightof irradiated larvae, whose pupation was delayed, was considerably
higher than that of the control larvae. In the present investigation, themechanism of this phenomenon has been studied in detail using several
organs and discs cultured in a synthetic medium after irradiation.
Four wild strains (Oregon-R, Canton-S, Kochi, and Samarkand) andseveral eye-color mutants (bw, lV, v, en, v bw, en bw, Bar, bar-3, andDpjIn(3L)P, In(3R)C, Sbel(3)e) of Drosophila melanogaster were used asmaterial. Third-instar larvae grown under sterile conditions were irradiated with various doses of X-rays (160 kVp, 25 rnA, 370 r/min with a
1 mm Al filter, at a distance of 30 cm.)
The larvae were dissected and various organs or tissues were taken out(cephalic complexes, wing discs, eye discs, leg discs, testes, ovaries,
salivary glands, fat bodies, and blood cells) and single cells from theseorgans or discs were isolated under a binocular microscope in a sterile
glass chamber.
The isolated cells were cultured in a normal synthetic medium, and in
the same medium containing a radioactive isotope (0.5 f1.c 32 P/ml and 1 f1.C
Thymine-HC/ml). The sensitivity of the cultured organs and cells toirradiation was determined by observing their growth and differentiation
and by measuring the amount of radioactive 32p and thymine-HC
incorporated into them.
When the irradiated eye discs, leg discs, and salivary glands were
cultured together with the normal cephalic complexes (from ten larvae),
they showed pronounced growth and differentiation. The incorporationof radioactive phosphorus into these discs or glands was comparable to* Department of Genetics, Faculty of Science, Osaka University.

RESEARCHES CARRIED OUT IN 1958

105

that in the non-irradiated discs or glands.
When the non-irradiated eye discs, leg discs, and salivary glands were
cultured together with the irradiated cephalic complexes (from ten larvae),
they showed pronounced growth, but no differentiation.
Since the cephalic complex is known to secrete a hormone controlling
growth and metamorphosis, it would appear that the normal activity of
the cephalic complex on differentiation of organs and discs is destroyed
after irradiation, even with low doses.
As mentioned above, the eye discs, the leg discs, and the salivary
glands were not affected even by irradiating with high doses, whereas
the wing discs and the ovaries were affected by low doses.
These results coincide with those showing the incorporation rate ofthymine-He into isolated single cells. The difference between the sensitivities of various organs and discs to irradiation could be elucidated
by the sensitivities of cells isolated from them.
As described above, the metamorphic hormone secreted from the cephaliccomplex was not affected, even after irradiation with high doses.
The results obtained in this experiment show that the delay in pupation and the decrease in emergence percentage seem to be due to damage ofthe cephalic complexes, especially of the ring glands, by irradiation. In
the bar-3 strain which, among the thirteen strains used in this experiment,
showed the highest sensitivity to whole body irradiation the cephaliccomplex seems to have a higher sensitivity to radiation damage than in
the other strains.

65.

UnsuccessfuL Attempt at in vitro Cultivation of Spermatogenic
Cells of the Silkworm*(By Toshihiko SADO)

An investigation of in vitro cultivation of spermatogenic cells of thesilkworm, Bomby.>; mori, was undertaken in order to explore a techniquewhich might be useful for the study of the sensitivity of germ-cells toradiation at successive developmental stages.
Spermatogonia and/or spermatocytes from early 5th instar larvae ofBomby:>; mori were cultured in blood medium prepared from larvae andpupae or the same species.
As was demonstrated by SCHMIDT and WILLIAMS (1953) in their tissue
culture of male sex cells of the Cecropia silkworm, growth and dif-

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

106

RESEARCHES CARRIED OUT IN 1958

ferentiation hormones are needed for the normal completion of spermatogenesis. Taking this fact into consideration, the blood of thefollowing stages was prepared as a culture medium: 3rd, 5th, and 7th
day of the 5th instar larvae, larvae at spinning, prepupae and midpupae.
In order to inhibit tyrosinase activity in the blood, either of the following
methods was used: i) adding a few crystals of phenylthiourea andcentrifuging, or ii) maintaining a temperature of 56°C for five minutes
and centrifuging. Though the latter method was superior to the former
in respect to the cleanness of the medium, the former was used as aroutine procedure throughout this experiment with a few exceptions,because heat treatment seemed to modify to a great extent the nature ofblood.
The individuals used were sterilized with 0.1 per cent aqueous solutionof mercuric chloride for about 30 minutes, washed in sterile tap water
three times, and then placed on a filter paper in a sterile Petri dish untildry.
A number of cysts containing spermatogonia or spermatocytes weresuspended in a hanging drop of the culture medium, and then incubatedat 27°C.
The attempts at cultivation have, however, been unsuccessful, showing
only degenerative figures. Fifteen to thirty hours after the suspension
was made, depending on the stage from which the blood medium was
prepared, a large number of minute bubbles appeared on the surface ofthe cysts. Then the cells gradually became abnormal and after five daysor more some follicles were broken off and the cells were liberated into
the surrounding medium.
As is well known, the physico-chemical nature of silkworm blood, i.e.,
pH, viscosity, osmotic pressure, and chemical constituents, etc., undergoes
considerable change with age at and after the fifth stage, owing to thedrastic change in the formation and in the secretion of silk substance;
during this period normal spermatogenesis proceeds. The same may betrue for the metamorphosing hormones.
It seems very difficult to control these dynamic changes in the properties of blood in vitro, although it may not be impossible.

66.

Influence of Radiation on Mitochondrial Function in
Cellular Metabolism*(By Saburo NAWA and Bungo SAKAGUCHI)

It is well known that various cytoplasmic granules are bounded by* This work was supported by Grant RF 57178 from the Rockefeller Foundation.

RESEARCHES CARRIED OUT IN 1958

107

semi-permeable membranes. The work to be reported here was initiatedin an attempt to evaluate the effect of radiation on the mitochondrial
function controlling the accessibility of the enzymes, substrates andcofactors.
The mitochondria were prepared from mouse liver and suspended in
0.25 M sucrose. The mitochondrial suspensions were put in ampoules,
kept in an ice-bath and exposed to varying doses of X-rays. After
irradiation, the activities of acid phosphatase and DNase II were measured
in both intact and fully activated mitochondria. Alternative freezing
and thawing was used for the activation of mitochondrial enzymes. The
results are shown in Tables 1 and 2.
Table 1.
Dose of X-rays(r)

Effect of X-rays on mitochondrial acid phosphatase activity.
Activity
-

-

Ratio

----

Total*

0.025M**

0.25M**

0.025MjTotal

100
95

82
80

65
58

82
86

90
85

85
83

58
51

95
97

0
10,00050,000100,000

0.25M/Total
65
62
66
61
--

*)
**)

-

----

Activation by freezing and thawing.
Final sucrose concentration of the reaction mixture.
Table 2.

Effect of X-rays on mitochondrial DNase II.

Dose of X-rays(r)- _ . - _ .. -0
10,00050,000100,000

Activity
---

Ratio

-

Total

0.25M

0. 25M/Total

100
110
132
128

27
29
54

27
27
41
60

-

-

77
-

- - - - - - - - -"--The ratios of acid phosphatase activity of intact mitochondria inisotonic sucrose to total activity were not markedly changed by increasing
dosages of X-rays. The total activity of acid phosphatase in activatedmitochondria slightly decreased in the course of a series of irradiations.
On the contrary, the ratios of activity of intact mitochondria in hypotonic
sucrose to total activity increased under these conditions.
The mitochondrial swelling (Table 3) caused by suspension in a hypotonic
medium, was measured by a decrease in optical density, and the degreeof swelling increased with an increasing dosage of X-rays. Therefore,

108

RESEARCHES CARRIED OUT IN 1958

the alteration of mitochondrial membranes seems to have been caused by
the X-rays.
Table 3.

Effect of X-rays on mitochondrial swelling.

Dose of X-rays(r)o
10,00050,000100,000

Decrease in optical density
(%)
100
116
150
176

In the case of DNase II, the activated mitochondria increased in
activity with increasing dosages of X-rays. The activation of DNase II
in mitochondria may be not completely achieved by this procedure but
the increased rates of activation were assumed to be caused by X-rays.
The activity of intact mitochondria increased obviously with increasing
dosages of X-rays. These results suggest that the reaction between
DNase II and DNA through mitochondrial membranes might be acceleratedby X-rays. In the cell, biochemical reactions are generally controlled by
the localization of the enzymes and substrates. It is probable that oneof the direct effects of X-rays is a removal of intracellular barriers
between enzyme and substrate. The damage may be intensified in the
many biochemical processes following, even though the actual change inthe subcellular particles is very slight.

67.

Studies on Genetic Variation in Response to Radiation in Mice
Effect of Single Doses of Total-body X-irradiation on the
Leukocyte Values of the PerijJheral Blood. *(1)(By Kiyosi TUTIKAWA, Masaaki ONOUE and Kotoyo TUTIKAWA)

In mammals, it has been shown that the blood-forming tissues areamong the most sensitive to ionizing radiations. After total-body exposure, manifestations of injury to the blood-forming tissues may appear
in the peripheral blood even in absence of detectable change in the bonemarrow or lymphatic tissues. In rabbits, it has been recognized thatlymphocyte values fall below the control level with doses of 25 r.
The present report deals with the peripheral blood changes producedby X-ray dosages ranging from 100-600 r.

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

RESEARCHES CARRIED OUT IN 1958

109

The mouse stocks used mainly in this study were SM/Rr and dba/Ms.
The former stock ,vas originated by selection for small body size andthese mice also have inherited leukopenia. In this experiment, the normal
leukocyte counts averaged 3418 in the SM/Rr strain and 7694 in the
dba/Ms strain.
Irradiation conditions were: at 160-kVp, 25mA with HVL of 0.3mm Cu
and 0.5 mm Al ; target distance 50 cm to the mid-point of the turn table.
Each irradiation group consisted of five animals which were placed on
the turn table and they were irradiated at 9 A.M. These procedures
were repeated in this experiment. Blood samples were obtained between
9 and 12 A.M. from the tails of mice 60 days old. We used CHAr's
method in which, before the blood was taken, the mice were placed in abattery jar and heated for approximately 5 to 8 minutes under a 60-\vattlight. The white blood cells were counted in the usual manner.

..

"''"
NI

dba/Ms

,.,
x

..
"
"

"
•
"
"
".
Time after exposurehr; hour,d: day,w: week
Fig. 1. Effect of total-body X-irradiation with 100-600 r
on the leukocyte values of the peripheral blood ofmice (strain: dba/Ms).

RESEARCHES CARRIED OUT IN 1958

110

The blood was examined for any change in leukocyte values at 1, 6, 12,
24 hours, 3 days, and 1 to 10 weeks, following single acute doses of 100,
200, 300, 400, 500 and 600 r. With all dosages as shown above, the total
leukocyte values of the peripheral blood reflected the rapidly changing
status during the first 24 hours after whole-body exposure. A mild
leukocytosis occurred within 6 hours and a maximum depression wasreached 3 days after irradiation (Fig. 1). The degree of this depression(No.X/o l )
75

..,

'5

~

~

A-

" ss

dba/Ms

V)

SM

0--

'"

U

.5

V)

W

f-

><.l

JS

0

:>:::
:::>

w

25

...J

IS

5
I

\

~ ----6._~.::---...

~:--.--

100

200

X-RAY

300

~oo

DOSAGE,

5QO

600

r

Fig. 2. Relation between X-ray dose and degree ofdepression of leukocyte value at 3 days after exposure in two strains of mice.

became more severe with increasing doses (Fig. 2). The leukocyte
values 3 days after exposure, however, were higher in SM/Rr than in
dba/Ms mice with dosages of 200 r or above, in spite of the fact that
normal mean counts in SM/Rr were lower than in dba/Ms mice. We
conducted, therefore, additional experiments using. six other inbred
strains or stocks with differential normal leukocyte numbers. However,no specific correlation with the normal leukocyte values could be seenfrom the counts of the leukopenic state.

RESEARCHES CARRIED OUT IN 1958

III

Recovery of the leukocyte value followed, and an absolute increase ora normal level was reached between the third and fourth weeks after
exposure of the mice to 100 r or more. The pattern of change in the
peripheral leukocytes following exposure is generally in agreement with
HENSHAW'S (1944) pioneering research.

68.

Studies on Genetic Variation in Response to Radiation in Mice
Strain Differences in Mice in the Response of Leukocyte
Values to X-irradiation*(II)(By Kiyosi TUTIKAWA and Kotoyo TUTIKAWA)

It has been previously reported that the maximum depression of peripheral leukocyte values was reached 3 days after a single total-body Xirradiation with doses ranging from 100 to 600 r, and no specific correlations with the normal leukocyte values could be seen from the counts of
the leukopenic state. During the course of this study, it was discoveredthat differences exist among strains of mice in the degree of maximum
depression of leukocytes 3 days after total-body exposure with doses of
500 r. This report presents the evidence on the genetic variation of thiscondition.
The mouse stocks used in this study were C57BL/6, dba/Ms, SM/Rr
and LG/Rr. Other inbred strains, BALB/c, C58/LwMs and DBA/2Jax,
were added in the course of this experiment. The irradiation conditions
and the procedures of blood sampling have been previously reported (I).
( 1) Age, sex and strain differences in the response of leukocyte values
to X-irradiation.
In a preliminary experiment using dbajMs mice 60 and 30 days old, anage difference was found; leukocyte counts in older and younger animals
were 179±67 and 111±52, respectively. Subsequently we used only mice
60 days old in this study.
The mean leukocyte values and their standard deviations under normal
conditions and 3 days after exposure to doses of 500 r are given for four
inbred strains in Table 1. The mean value of the leukocytes was computed by pooling the count in each strain, since an insignificant difference
between sexes was found except in the case of strain C57BLj6. To test
whether the variation in leukocyte value is normal within a strain, thedata were plotted on probability scale paper, and the points fell along a
straight line. From the magnitudes of the standard deviations, it can beseen that the variation in leukocyte values seems to depend upon themean counts.
-* This work was supported by Grant RF 57178 from the Rockefeller Foundation.

112

RESEARCHES CARRIED OUT IN 1958
Table 1. Mean counts of leukocytes, in normal condition and 3 days
after X-irradiation with doses of 500 r in four strains.
Total number of leukocytes, cells/mm)----

Number ofmice

Strain50
42

C57BL/6
dba/Ms
SM/Rr
LG/Rr

50
50

Normal--_._-----

3 days

Mean

If

Mean

8598
6449
3876
5802

2009
2409

177
179

985
1553

372
550

If

61
67
102
166

The difference in mean counts of leukocytes 3 days after exposure todoses of 500 r between strains C57BL/6, SM/Rr and LG/Rr is statisticallysignificant, but the difference between C57BL/6 and dba/Ms is insignificant.
The results of the present investigation lead, therefore, to the conclusion that strain differences can be seen in the response of leukocyte values
to radiation.
( 2) Genetic analysis of radiation response to leukocyte values
Three inbred strains, dba/Ms, SM/Rr and LG/Rr, which show different
radiation responses in leukocyte values, were used for further genetic
analysis of this sort of response. This report presents only the results
obtained from six genotypic groups including two inbred strains andtheir hybrids. Maternal influence within each hybrid group was roughlybalanced by making reciprocal matings between the two parental genotypes.
Table 2. Means and standard deviations of leukocyte values, in normal
condition and 3 days after X-irradiation with doses of 500 r ineach genotypic group of mice.
Total number of leukocytes, cells/mm 3
Number of
mice

Generation- Ps (SM/~Normal

3 days

Mean

I

I---soPL (LG/Rr)

50

F1

~

F2
Bs
BL

108
48
40

Mean

3876
5802
5152
4756
3830

985
1553
1553
1456
1255

5095

1640

372
550

486
437
297
505

102
166
127
180
114
138

RESEARCHES CARRIED OUT IN 1958

113

The mean leukocyte values of each genotypic group and their standard
deviations are given in Table 2 for normal mice and those tested 3 days
after exposure to doses of 500 r. The leukocyte values of the F I and F 2
generations in normal condition and in the leukopenic state were closer
to those found in PL(LGjRr) than in Ps(SMjRr). The values of the
backcrosses to SMjRr; Bs were shown to be approaching in magnitude
those of the P s , and the values of the backcrosses to LGjRr; B L wereshown to be approaching those of the PL. The rate of approach in theBs , however, was slightly higher than that in the B L. These results arein agreement with CHAr's study (1957) on the leukocyte values of inherited leukopenia in normal conditions. The present evidence indicatesthat radiation response, as shown by the magnitude of the leukocyte
value 3 days after exposure, is genetically controlled and possibly determined by a small number of genetic factors.
The mean counts of F I and F 2 mice 3 days after exposure were quite
similar, but the variance of the F 2 was the largest among six genotypicgroups. Assuming that the variance of the F I is an estimate of theenvironmental variation and that the variance of the F 2 is environmental
variation confounded with genetic, their variances may be used to estimate
the relative magnitudes of the effects of genotype and environment. As
an attempt to estimate the genetic contribution to the variation in theF 2 generation, analysis of variance was made for the data obtained fromthese mice (Table 3). In the present experiment, the magnitude of theheritability of the response of the leukocyte value to radiation was roughly
estimated as 0.809 in the F 2 generation.

Table 3.

Analysis of variance of response of leukocyte values to radiationin the F 2 generation.

Source of variation

Sum ofsquaresd. f.

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

Total
Between litters
Within litters107

I 1377.76

10I 606.20

97I

771. 56** Significant at the 1 % level.
di'=13.36,

Meansquarei

Expectation ofmean square--,-------------- - - -60.62**
7.95

I

dE? +1/2d02+~du2<1!?+1/2 lfo 2

114

RESEARCHES CARRIED OUT IN 1958

1.

RADIATION GENETICS IN PLANTS
69.

Radiosensitivity in Plants(By Tar6 FUJII)(A) Experiments with cultivated plants
Dry dormant seeds of 43 species or their commercial varieties were
irradiated by r-rays from radiocobalt and the radiosensitivity of theseplants was determined by measuring the decrease in rate of germinationand in number of growing plants. The germination rate decreased with
the increase of dosage, but it varied widely, not only between different
species but also between different commercial varieties. An irradiation
effect was also noticeable in the delay of seedling growth proportionate
to the dosage, as well as dwarfing, asymmetrical development anddestruction of venation in young leaves. Hordeum was the most resistantand Triticum was the most sensitive among the Gramineae The Liliaceae were the most sensitive and next to them were the Umbelliferae.
In some species of Cruciferae, Leguminosae and Compositae a few plants
continued to grow even when irradiated at 100 kr. They were, therefore,
more resistant to radiation than the Gramineae, Commelinaceae andLiliaceae. From our experiments, LD-50 for seed irradiation was about
as follows:

Hordeum sativum
Triticum vulgare
Sorghum bicolor
Zea maysRaphanus sativus
Arachis hypogaea

70 kr
20-40
40
/I

70
/I

Glycine max
40-70 kr
Vigna sinensis
Pharbitis Nil
70
Solanum gilo
20-40
Calendula arvensis
70
Cosmos bipinnatus 70-100/I

The germination and growth up to the appearance of the cotyledons
was not much decreased in several species, even at 100 kr, but most of
the seedlings died immediately after germination and only a few plants
could survive. Knowing this, the sensitivity of dormant seeds to irradiation can be determined by finding the percentage of seedlings whichdevelop 2-3 leaves. Radiosensitivity was different even between commercial varieties within the same species. It seems that radiosensitivityof plants may be due to the genetic make-up of a race, and it may be
modified by physiological or physical conditions as well.
(B) Radiosensitivity of mutants in Einkorn wheat
Five mutants of Triticum monococcum flavescens induced by X-rays were

RE:EARCHES CARRIED OUT IN 1958

115

irradiated again with X- and r-rays, and the differences in radiosensitivity
were examined with respect to the recessive genes induced by mutation.
Three kinds of chlorophyll mutants and two morphological mutants
were used in this study. Dry dormant seeds were irradiated by X-rays
at 10, 20 kr and r-rays at 20 kr, and the germination rate, plant height
and fertility in the Xl and frequency of chlorophyll mutation in the X ewere examined (Table 1). The decreases in germination rate, survival
rate and fertility of the Xl mutants were not much different from thoseof the normals. But the chlorophyll mutation rate seemed to have become
Table 1.
Genetic effects of radiation in mutant strains of Einkorn wheat.

StrainI
I

Dosage(kr)

Normal

0

'

X-lO

X-20
r-20
Chlorina

Basi-viridis II

I

0
X-I0
X-20
r 20

37.5
50.0
45.0
37.5

0

30.0
30.0
50.0
26.7

X-20
r- 20

o

60.0
62.5
47.5
47.5

X-I0
X-20
r-20
Early

o
X-I0
X-20
r- 20

Slender

65.0
70.0
62.5
75.0

X-lO

Virido-albina

Germination rate
(%)

o
X-I0
X-20
r- 20

I

Plant
height
(em)
11.8

12.0
8.5
8.6
10.2
9.0

7.7
4.9
9.6
11.8

8.4
9.2

Frequency % of nonchloro.'
X1 f ertl'1'Ity ofh 11 muta- ~ermmat(%),.Pt.Y·IOn In X ., I mg head.
(~)". progemes
79.3
79.5

45.0
41.6
54.9
53.7
37.4
28.8

0.0

4.6
0.0

60.6
0.0

3.3
0.0

0.0

16.7
26.8
56.5
69.2

51.5
46.9
48.4

0.0
0.0
0.0

23.9

0.0

47.8
25.0

76.1
81.1

0.0

0.0

5.0

2.4
0.0

9.1

9.8

10.1
8.8

6.4

55.0
40.0
20.5
17.5

12.2

3.5

13.7

0.0

87.5
75.0
55.0

11.4

62.0
54.4
34.6
41.0

0.0
0.0
0.0

55.0

8.5
25.0
16.1

10 .1

4.3

9.8

7.5
7.1

0.0
37.7
63.1
46.7

116

RESEARCHES CARRIED OUT IN 1958

slightly lower than that of the normals, while the frequency of nongerminating head-progenies in the X 2 was higher in the mutants than in
the normals.
Thus we see that radiosensitivity is not different in theexamined mutants in comparison with the normals. A decrease in
viability in the X 2 of the mutants as shown in an increase of non-germinating head-progenies, may be due to an accumulation of genic mutations
and radiation damage.
In this study, back mutation from recessive to dominant, such as fromchlorina to normal, did not occur.
(C) Experiment with cultivated rice
A preliminary irradiation experiment was carried out in 1957 for the
purpose of determining the lethal doses for rice. Dosages were 20, 40,
70 and 100 kr by r-rays from radiocobalt, and they were applied to twocommercial varieties, Omachi and Tetep. The germination rate in bothvarieties did not decrease greatly up to 40 kr, but it was very low at
70 kr. In 1958, three varieties, Tomoemasari (a typical Hokkaido variety),
Mihonishiki (Chubu district) and Norin No. 18 (Kyushu district) were
irradiated with 20, 40, 50, 60 and 70 kr. With regard to germination
Table 2.
Effects of radiations in three varieties of Oryza sativa.

II Plant height(em)
I

Dosage(kr)

Germination
rate (%)

0
20
40
50
60
70

79.5
81.0
67.5
67.0
1.0
0.0

15.03
13.83
12.06
11.05

Mihonishiki

0
20
40
50
70

80.5
82.0
55.0
18.5
0.0

11.83
10.50
6.80
6.13

Norin No. 18

0
20
40
50
60
70

86.0
81.0
26.5
13.0
0.0
0.0

10.83
10.51
6.82
5.20

Variety
Tomoemasari------_.----

I

I
I

I

I

Survival rate
(%)

77.0
78.5
65.0
61.5
0.0
0.0
77.0
77.5
39.0
13.5
0.0
85.5
81.0
25.5
15.0
0.0
0.0

RESEARCHES CARRIED OUT IN 1958

117

rate, plant height and survival rate, Tomoemasari was most resistant,while Norin No. 18 was most sensitive (Table 2). Fertility in the Xl
generation was examined in Mihonishiki; it decreased with the increaseof dosage, from 90.69.6 in the non-irradiated group to 55.3% at 20 kr, andto aoout 20% at 40 and 50 kr irradiation. A similar examination was
carried out with several other varieties and a few wild rice species.
From these results, it seems that the LD-50 of cultivated rice is about
30-40 kr, and 10-20 kr irradiation given to dormant seeds would be thevroper doses for mutation work with rice.

70. Differences in Radio-sensitivity and -mutability between
Cultivated and Wild Strains of Japanese lvIorning Glory(By H. KIHARA and S. SAKAMOTO)

In order to compare the differences in radio-sensitivity and -mutability
between cultivated and wild strains of Japanese morning glory (Pharbitis
nil Chois.), the following four strains were used in this experiment. (A)wild strain "850" -collected in Nepal with standard leaf and blue flower,(B) wild strain "Tendan" -collected in North China with standard leaf
and blue flower, (C) cultivated strain "Violet" -with dragon-fly leaf andreddish purple flower, (D) cultivated strain "P7"-with standard leaf andblue flower. Dormant seeds were irradiated with r-rays from a BOCO
source. The seed-coats were cut open and the seeds were sown.
Table 1. Percentage of germinating and surviving plants in 3 strains.
Strain

(C)

(B)

(D)
-~

Dosage I No. of Germi- Survival(kr) , sowing nation_ . _ - ~ - -

Cant.
10
20
30
40
50
60
70
80
90
100

46
50
50
48
50
50
50
50
50
50
50

- - _ .. _-

100
100
98.0
97.9
98.0
96.0
100
100
96.0
100
96.0

----------

100
100
95.9
100
89.8
79.2
44.0
48.0
27.1
22.0
8.3

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

__ ..--

No. of Germi- Survi- I No .. of Germi- Survival
sowing nationval SOWIng nation
49
50
50
50
50
50
50
50
50
50
50

100
96.0
92.0
92.0
100
96.0
96.0
98.0
94.0
100
98.0

95.5 I97.9
100 I
95.7 I
82.0
66.7
43.8
38.8
4.3
0
0

49
50
50
50
50
50
50
50
50
50
50

100
100
98.0
100
100
100
100
100
100
98.0
98.0

-

--

100
100
95.9
96.0
60.0
28.0
26.0
28.0
4.0
0
0

118

RESEARCHES CARRIED OUT IN 1958

(I) Difference in radiosensitivity
Table 1 shows the percentage of germinating and surviving plants ill
3 strains, (B), (C) and (D), after irradiation at dosages of 10 to 100 kr.
Germination and survival were observed 5 and 45 days after sowing, respectively. The germination of the 3 strains was very good and uniform
at any dosage. The number of surviving plants was high under 40 kr
but at dosages over 50 kr it decreased rapidly with the increasing dosage.
All plants of the cultivated strains (C) and (D) were dead at 90 kr ormore. The wild strain (B) showed a higher survival rate than the cultivated strains at all dosages. In this experiment, radiosensitivity of thecultivated strains was higher than in the wild ones. Several abnormalities, such as irregular leaf, shrunken leaf, variegated leaf, blocked
hypocotyl elongation, and abnormal anthocyanin formation in hypocotyl
and cotyledons were observed.
(II) Difference in radiomutability
In this experiment dormant seeds of (A), (B) and (D) strains weretreated with 25 kr. Radiomutability was measured by the frequency of
induced mutations. 330 X 2-plants derived from 22 Xl-plants of (A) wereobserved but no visible mutation could be found. Among 105 X2-plants
Table 2.

Pollen- and seed-fertilities of delicate leaf mutants on the "P7"
strain. (No. of flowers in parentheses)
Seed-fertility (%)*

Culture numberI
36-1***-5-31

Pollen-fertility
(%)
6.06
12.05
41.03
21.93

37-6
-7
-10

27.07
73.85

-21
Control

25.72
90.51

selfedbackcrossed**o (Many)
0

(17)
(7)
0
2.67 (61)(79)
00.83 (37)(19)
081.00 (43)

5.50
(1)
0
4.67 (18)
11.16 (9)11.83 (7)2.83 (12)open pollination
ca. 0.80 (ca. 40)
8.17 (65)(28)
05.00 (230)
10.33 (69)
5.17 (152)0.83 (38)71.33 (57)

* Seed-fertility=(No. of seeds / No. of ovules) x 100(No. of ovules=6 x No. of flowers)** Backcrossed to control.
*** Did not form elongated branches.

obtained from 36 Xl-plants of (B) only one mutant with deep purple
flowers was observed but there was no difference from the control plants
in the other characters. Out of 912 X 2 -plants obtained from 43 Xl-plants

RESEARCHES CARRIED OUT IN 1958

119

of (m, 9 mutants were found. One was a maple leaf (Tatsutaba) mutant
with high seed-fertility which could be easily backcrossed to the control.
The other 8 individuals were delicate leaf (Sasaba) mutants. Their
growth was slow and dwarflike but later they formed elongated branches.
Their flowers with slightly separated petals did not fully open and thedevelopment of the stamens was abnormal. Table 2 shows pollen- andseed-fertilities of the delicate leaf mutants. They were both very low.
In this experiment the frequency of induced mutations in the cultivatedstrains was higher than in the wild ones.

71.

Relation of Ploidy to Radiation E)Ject(By Mitsuya NEZU)

The effect of radiation is influenced by various factors.
T:lb!e l.

Polyploidy is

Frequency of chromosome aberrations at MI in PMC's induced
by r·rays in wheats.
Total number observed
Total no.
- Average no.
I
mu!tivalents ! of breaksof cells
frag·univaobserved! ments
per cells
lents !
eli})
1(4)
GD
®
I
I
---,------.-;\Iateria!

Dosage
(kr)

-_.",

I,

T. aC[JilopoirLcs

Cont.

20
T. 1nonOCOCCU1n
T. d~coccwn
T. dnrumT.!SpclIa

T. vulgare

4
4

Cant.

6

20

3'J

Cant.

7
19
9
7
27
27
27
17
26
18
9
6
27
16
14

20
30
Cant.

20
30
40
Cant.

20
30
40
Cant.

20
30
40

2 1
!

1
7

10

1
4

12 3
14

2
3
2

12 1
22 2
30 2

6

27 3
28 8
11 3
1
21 5
17 6
15 4

I

2
1
1

6

6
2
2
11

3
- - - - - - - _ .. _-

I

5

2
1

1
2

1

4

2

0.00
1.75
0.00
0.66
0.14
2.15
3.55
0.00
1.07
1. 96
2.51
0.00
2.73
5.38
6.88
0.66
2.77
4.43
5.07

120

RESEARCHES CARRIED OUT IN 1958

one of them. The author has examined in this connection meiotic
irregularities and seed fertility in three groups-di-, tetra- and hexaploid-of wheat. As material 6 species were used, namely Triticum a"gilopoides, T. monococcum, T. dicoccllm, T. durum, T. Spelta and T. vulgar,'.
Dormant seeds were exposed to r-rays of 20, 30 or 40 kr for 18 hours.
The percentage of PMC's with meiotic irregularities, mostly reciprocaltranslocations, increased with increasing ploidy. In 4x species the great
majority (83.4%) of PMC's showed aberrations at 30 kr and in 6x species
88.7% cells showed similar irregularities at 20 kr, while in 2x species only
38.3% had aberrations at 20 kr. At higher dosages (30-40 kr) all cells wereabnormal. A similar increase of multivalents (@, @, ([0) was observed,
especially in 6x species (Table 1). As to other abnormalities, asynapsis
and deficiency were observed, and rarely heteroploidy. The number ofbreaks per cell increased almost linearly with the dosage within eachgroup. Comparison between the three groups-2x, 4x and 6x-showed anincrease in breakages in a ratio of 1 : 2 : 4. Seed fertility decreased withincreasing dosage. The 2x species were most susceptible in this respect,
next to them the 6x, followed by the 4x. The fertility of the 6x species
decreased suddenly at dosages between 20 and 30 kr. The higher theploidy, the more aberrations arise. The highest number of multivalents
was found in the 6x plants.

72.

Genetic Effects of {3- and r-radiation on Einkorn Wheat(By Seiji MATSUMURA)

To compare the genetic effects of {3-radiation with those of r-radiation,
seeds of Triticum monococcum jlavescens were soaked in :>2p and 131 1
solutions for 2 days just before sowing. Radioactive solutions of pH 6
to 7 contained 0.05~0.4 mc/gm of 32p and 0.2~O.8 mc/gm of wI. Also rirradiation with GOCo was applied at the dosages 2.5, 5, 10 and 20 kr
immediately after soaking the seeds in water for 2 days. The growth ofseedlings, single-spike fertility and chromosome aberrations of treatedplants (Xl) and the chlorophyll mutations in the X 2 were compared for{3- and y-irradiations. The data are shown in Table 1. The seedlings
were measured 18 days after sowing. The relation between the inhibitionof seedling growth and dosage of {3- and r-radiations coincides roughlywith that between the decrease of fertility and dosage. r-rays at 2.5 kr
slightly inhibited the growth of seedlings and reduced the fertility andthe effects correspond roughly to those of {3-radiation from 0.2 mc/gm 32p
solution and 0.8 mc/gm 1311 solution. r-irradiation at 10 kr was markedly

121

RESEARCHES CARRIED OUT IN 1958
Table 1.

DosageControl

r-ray

j'' C
5.0 f!
10.0 f!
,20.0 f!

Genetic effects of (3- and r-radiations in Einkorn wheat.
Germination
rate
(%)

Length ofseedlings*(em)Fertility
in Xl
(%)

38.0
42.0

13.46
10.12

92.09
83.17

56.0
16.0

9.10
2.71

64.63
47.36

14.33
13.13
12.70

92.25
89.02

54.0
76.0
0.2 f!
48.0\ 0.4 f!
68.0
f
0.2
me/gm
1311
l 0.8 f!
44.0

I

0.1

Chlorophyllmutationsin X 2
(%)

0.00

0.00
14.00

2.44
24.53
90.91

17.46
28.57

6.89
1.35
7.69
7.94

5.19
5.33
11.11
18.87

2.13

0.00
6.45

0.0

f 0.05me/gm 52.0
22p

Chromosome
aberrationsin PMC
(%)

"

7.97
12.46
11.63

82.25
71.18
95.00
88.38

O.OJ

* The seedlings were measured 18 days after sowing.

effective. There was no germination at 20 kr with r-irradiation.
In the treated plants (Xl), white and yellow stripes were often found,especially after 32P. The frequency of ears with chromosome aberrations
in Xl-plants was strikingly high after r-irradiation. The majority of
induced chromosome aberrations were: ®+5n, often ®+®+3n or ®+4n
and seldom @+3n or asynapic 14,.
The frequency of head progenies with chlorophyll mutations in the X 2 generation increased with the increase of radiation dosage. The effectsof J3-radiation from 0.4 mc/gm 32p solution correspond roughly to those of
5.0 kr r-radiation. The majority of chlorophyll mutations after J3-irradiation were albina, xantha and viridis, while with r-irradiation alba-viridis,
virida-albina and basi-viridis were most often observed.
In conclusion, the effects of J3-radiation from 0.2mc/gm 32p solution and
0.8 mc/gm 131 1 solution, correspond to those of 2.5 kr r-radiation. If weassume that the effects of J3-radiation are confined to the embryo, by calculation we find that the 0.2 mc/gm 32p solution equals about 2.4 krad.
This will account for the present data.

73.

Genptic Effects of Thermal Neutrons on Wheat(By Seiji MATSUMURA)

The thermal neutron irradiations discussed here were conducted in the

122

RESEARCHES CARRIED OUT IN 1958

thermal column of the Japan Atomic Energy Research Institute's Nuclear
Reactor, JRR-l. Hole No.7 (North 6// if> Thermal Column Access Port)
was selected to keep gamma contamination as small as possible. The
thermal neutron flux was calculated to be 2.6 x lOR n/cm 2 • sec when thereactor was operated at 25 kilowatts.
In a preliminary experiment, dormant seeds of Triticum monococcum
flavescens were kept in this hole for 1~5 weeks (I~ V: actually 131.5~
1068.7kWh: 4.9~40.0x1012n/cm2). The data are shown in Table 1. The
higher the dosage of thermal neutrons, the more delayed were the germination and growth of seedlings. The seeds were almost uniformly
injured. There was no germination after a 4~5 week treatment (lV~V:
31.4~40.0x 1012 n/cm") and the seedlings resulting after shorter exposure
died in an early stage. In the root tips just after germination thefrequency of chromosomal aberrations increased with the increase indosage, and in IV and V more than 95% of the dividing cells had
chromosome aberrations. In PMC's a ring of 4 chromosomes was observed.
In the X 2 , chlorophyll mutations such as albina, xantha, viridis, etc., were
found. The frequency of chromosome aberrations and chlorophyll mutations increased in proportion to the dosage.
In the next experiment the reactor was operated at 40 kW and seeds of
T. monococcum (2x), T. durum (4x) , and T. vulgare (6x) were subjectedto thermal neutrons at 5 different distances in reactor hole No.7 for 2
weeks (actually 990.8 kWh). The thermal neutron flux ranged from 0.49
to 4.2 x 10~ n/cm 2. sec (total flux: 4.4~37.5 x 1012 n/cm 2). After exposure to
Table 1.

Genetic effects of thermal neutrons on Einkorn wheat.

ThermalKilowatt ne~~;nhours( x 1012
n!cm")
Control
I
II
III
IV

V
*

131.5
363.2
491.5
838.0
1068.7

4.9
13.6
18.4
31.4
40.0

Germination
rate*
(%)

65.0
65.0
79.0
87.5
20.0
40.0

Lengthofseedlings(em)Abnor- Chromo- Chlarophyll
mal cells some(Index) in root aberra- mutations in
tions
tips
PMC's
in X2
(96)

9.41
7.76
7.08
4.86
0.50

(100)
(82.47)(75.24)(51. 65)( 5.31)( 4.99)

0.47

0.24
22.04
28.30
4:3.67
96.43
96.61

(%)

( Yo)

O.CO

0.00
3.77
10.53
8.00

3.13
18.52
15.79

The seedlings were measured 14 days after sowing.

37.5x1012 n/cm 2 , T. monococcum did not germinate, while the seeds of T.
durum and T. vulgare germinated but the seedlings died in an early stage
without further growth. At 20.6 x 1012 n/cm 2 about 2/3 of the seeds of T.

RESEARCHES CARRIED OUT IN 1958

123

monococcum germinated, but the seedlings soon died, while in T. durum
and T. vulgare slow growth of the seedlings was observed. Thus we seethat T. monococcum is most sensitive to thermal neutrons and T. durum
is unexpectedly most resistant. There is no significant difference between
T. durum and T. vulgare.
74.

Genetical Analysis of a Few Chlorophyll iVlutants inEinkorn Wheat Induced by Radiation(By Taro FUJII)

Crossing experiments between basi-viridis II and other mutant
strains
Crosses between basi-viridis II and basi-viridis I or other mutant strains
were carried out in 1957-'58, and double recessive segregants wereobtained from each cross. Basi-viridis II and basi-viridis I recovered anormal chlorophyll content when they were placed in the phytotron, but
in the seedling stage the chlorophyll content was only about 5096 in comparison with that of the normals. The chlorophyll content of the double
recessives derived from the cross basi-viridis I X II was further decreased
and the plants had the appearance of virido-albina mutants. When they
were placed in the phytotron, their leaves gradually recovered a normal
green color, as did both parents. On the other hand, early, slender andirregular-ear mutants were crossed to basi-viridis II and double recessiveplants were also obtained from these crosses. As to chlorophyll, alldouble recessive segregants in the seedling stage were similar to the basiviridis II parent.
From these results and similar symptoms exhibited by double recessiveplants obtained from crosses between virido-albina and the chlorophyllmutants (Ann. Rep. No.8), it seems that the chlorophyll content of the
double recessives in the seedling stage is decreased more than in theparents. On the contrary, the chlorophyll content and recovery in double
recessive plants obtained from crosses between chlorophyll mutants andmutants of other kinds were quite similar to those of the parental
chlorophyll mutant, and they resembled morphologically the other parental
mutant. This may be due to the fact that in the crosses between chlorophyll mutants the genes involved have an additive action causing adecrease of chlorophyll content, while the genes for morphological traits
have no effect on chlorophyll formation.
(A)(B) Doubly recessive plants from crosses between chlorina and striata
All F 1 plants from a cross between chlorina and striata were of normal

124

RESEARCHES CARRIED OUT IN 1958

green color and showed at meiosis the chromosome conjugation lrv+5n
while in both parents seven normal bivalents were formed. Normal,
chlorina, striata, and doubly recessive plants segregated in the F 2 generation. The segregation ratio was also observed in the F 3 generation, but
the linkage relationship was not clear because the germination rateof chlorina was low, and that of striata was still worse. In the Fa
striata (or chlorina-striata) plants were segregated only from the F 2 plants
with a tetravalent (Table). Moreover, F I plants from crosses between
striata and the mutants basi-viridis I or II, slender and early also showed
the conjugation 1,v+5n. From these facts, we judge that the striata
gene must be located on one of the chromosomes involved in the reciprocaltranslocation.
:

Character in F 2 I

I

Chromosome
conjugation
in F 2

711

"

"
"

Normal"Chlorina

75.

------_.,

chlorina

normalstriataI

NormalChlorina

Segregation rate in F 2--------------,

1n~ +5rr

"

98
141
207
176

"

!

chlorinastriata---"'-----

46
147
67
201

34
21

8

35

Radiation-induced Mutation of the Self-incornpatibility
Gene in Brassica(By S. S. RAJ AN)

The present note is a preliminary report of attempts to induce mutations of the self-incompatibility gene in some species of Brassica. Maturepollen grains and flower buds at various stages of meiosis were subjected
to X- and gamma-radiations with doses ranging from 200 to 3,000 r units.
The pollens treated was used in self-pollinations. The gamma-ray treatments were confined to pollen grains only. Even the highest dose did
not affect the normal functioning of the pollen grains as checked by test
crosses with cross-compatible genotypes. It was noticed that neither ofthe radiations had any mutational effect when applied to mature pollen
grains. There was, however, considerable stimulation in the developmentof fruits, mostly with very few seeds. True seeds with 100% viability
were found after self-pollination with pollen from flowers opening on the
fourth day after irradiation, and later. There was a steady decline in

RESEARCHES CARRIED OUT I~ 19,,8125

the seed set as buds treated at younger stages came to flower and wereused in pollination. This is probably due to the high sensitivity of theearlier stages of meiosis to the radiation. Buds that were at early prophase to anaphase I stages during irradiation produced no fertile pollen.
Considering each seeds set as due to the mutation of a single selfincompatibility gene, the mutation and the dosage showed a linear relationship, being maximum (average of 5 seeds out of 10 ovules per frui(
at 500 r units and minimum (1 seed) at 220 r units, (control 0 seeds per
fruit). If these are true mutations, the fact that they could be induced
at the tetrad stage lends further support to the hypothesis that the pollen
reaction in these materials is under sporophytic control. Alternative
explanations for the seed setting might be (i) pseudo-fertility, (ii) mutation of modifiers, specific or nonspecific in reaction, (iii) physiologicalweakening or destruction of the incompatibility reaction without genetic
consequences. These have to be examined by results from test crosses
with genotypes homozygous for the S alleles. This work is in progress.

J.

GENETICS AND CYTOLOGY OF TUMORS

76.

An Attempt to Explain the Origin of Tumor Cells
with V- or j-elements(By Tosihide H. YOSIDA;

Tumor stem cells in almost all transplantable rat tumors contain prominently large V- or J-shaped elements. According to MAKINO andSASAKI (1958), the original stock of the Yoshida sarcoma of the rat contained a large V-shaped element in the tumor stem-cells, but threetumor sublines which were characterized by containing two, three andfour large V-shaped elements have been obtained from the original stock
tumor. Recently a new subline which contains two large V- and one
large J-shaped element was derived from the Yoshida sarcoma CYoslDA
1958, 1959).
On the other hand, some primary rat hepatomas which developed afteradministration of an azo dye were observed by YOSIDA 1957, and YOSIDA
and ISHIHARA 1958. According to their reports, some of the hepatomas
primarily developed lacked such large V- and J-shaped elements asobserved in the transplantable rat tumors, while other hepatomas werecharacterized by containing a large V- or J-shaped element. The frequency of the occurrence of the cells containing these elements varied withthe tumor.

RESEARCHES CARRIED OUT IN 1953

126

Based on the above investigations, a scheme representing the development of tumor cells containing large V- or J-shaped elements is given in
Fig. 1. Developmental events occurring in the hepatomas and the Yoshidasarcoma are represented on the left side and the explanations for theseoccurrences are given on the right side of the diagram. The process
going on in the cells is tentatively termed "primary SOIl1atic mutation".
The original hepatoma cells which do not contain a large V- or ]-shaped
element may have undergone this change. In the course of successive
cell generations a secondary somatic mutation may occur. Hepatomas
which are characterized by tumor cells containing large V-shaped
elements may belong to this category. If the tumor cells thus developed
are serially transplanted into other rats, the selection process which takes
place may determine which of the cell types will persist in the population. In this way, a stem line of the transplantable tumor could be
NORMAL 'HEPATIC CELls{(AOMIN1STR4T~OF}

HEPATOMA CELL OCCURRED

f

CELLS

}

\

V-CHROM.

l

PR!w.RY SOMATIC htUi1I.TICNj"

GROWTH OF HEPATOMA~~~~~~~

v'

V-CtltRO~SERIAL 'TRANSPLATATiON - ( I

I
I

v

v v v

I

I

v
SERIAl.. TRA1'fSPLANTATIOO { II

SERIAL TRANSPLANT;.,nG*t

V

v

I

I

TUMOR

TRANSPt
TUMOR

r.lUTATED CEllS

v

v
I

>

! S;:1N~~;'": I :}TE=~~~:':~rl'RY
I"

5UBUNEv

J~~~~~~

CELLS~~~~~ O£VELOPME~~~:~bO{M:::v

CEI,.LS

GROWTH OFSECONDARY MUTATED

o~g~~~bA ;~~~. v v
F

Gr.OWTH OF MUTA7ED}SE~~~~~'EiOMAT'C

OCCURR{XGROWTH OF. HEPATOMACELLS CO~TAININGNEWI

NO~MAL SOMATIC CELLS

Aza DYE)

v
I

I

•

I

I

I

I

I

v
I

I}

SUBUNEOF li~@G>~~~@l~~~~ciNEW

SER&AL'1'1V.HSPLANTATQt
5UBUNEYQSHOA SARCOMA

Fig. 1. A diagram representing the author's concept of the developmentof tumor cells containing the characteristic V- or J-shaped element in thecourse of cell generations. Thin circles denote normal somatic cells.
Heavy circles show the tumor cells. V and J inside the circle~ denote thecharacteristic V- and J -shape chromosomes of tumor cells.

established. The stemline karyotype may readily be altered by ensuing
somatic mutations. The new subline of the Yoshida sarcoma which con-

RESEARCHES CARRIED OUT IN 1958

127

tains a large J-element is a good example of a tertiary somatic mutation.
This study was reported on August 23, 1958 at X International Congress of Genetics, Canada.
77.

Chromosome Breaks in Yoshida Sarcoma Cells Inducedby Xirradiation*(By Toshihide TABATA, Hiroyuki HIRUMI and Tosihide H. YOSIDA)

The tumor stem cells of the Yoshida sarcoma used in this experimentare characterized by 40 chromosomes, among them two large V-shaped
elements. Rats carrying the Yoshida sarcoma were irradiated by X-rays
under the following conditions: Deep therapy installation 160 kVp;
filter of 1.0 mm AI; five different dosages, namely 10,000r (25.9 minutes),
5,000 r (17.9 minutes), 2,000 r (5.1 minutes), 1,000 r (2.5 minutes) and 500 r(1.7 minutes). The chromosomes of the tumor cells were prepared by theacetic orcein squash technique immediately after irradiation. The resultsof our observations are as follows:(1) Remarkable chromosome breaks were induced by X-ray irradiation
with high doses such as 10,000 r, 5,000 r, 2,000 rand 1,000 r, but the cells
were unsuitable for cytological analysis because the chromosome breaks
were too numerous to study. The X-ray dose most suitable for chromosome observation was 500 r.
(2) Idiograms of thirty four tumor stem cells containing chromosomal
breaks were analysed. The chromosomes of the Yoshida sarcoma comprise three types, namely ]-, V- and rod-shaped chromosomes. As astandard for measurement of each chromosome the mean value of thelargest V-shaped chromosome measured from thirty-four stem cells wasused. Chromosomes of stem cells were classified into nine different
groups, 0.2, 0.3, 0.4,-1.0 according to their a-ratio (mean length ofchromosome/standard length) (Table 1). As shown in the table, thefrequencies of breaks in the rod chromosomes were higher than in theother two groups. The x2 test shows that there is a significant difference
between the frequency of breaks in the rod-chromosomes and in the Vshaped ones.
(3) The locations of breaks are represented by a calculated value off/a (a=length from the kinetochore to the tip of the chromosome; f=length from the kinetochore to the location of the break). Most chromosome breaks were in the middle of the chromosome arms.
It may be concluded that the rod-chromosomes, especially those having* This work was supported by Grant RF 57178 from the Rockefeller Foundation.

128

RESEARCHES CARRIED OUT IN 1958

an a ratio of 0.8, are most sensitive to X-ray irradiation, and that theradiosensitive region is close to the middle of the chromosome arms.
Table 1.

Frequencies of breaks i'1 J-, V- and rod-shaped chromosomes.J -shaped chromo

n~

ratio

nl anJ

0.1
0.2
0.3

0
6
10

0.4

4

0.5

4
2

1.6
2.0
1.2

0.6

o
1.2
3.0

Rod-shaped chrom_

V-shaped chromo

fJ fJ/,mJ

nV

o
o
o

(I

0

o

3

1.8 I

2

8
7
12

1.0
5.8

o
o

3.7

0
0
0
2

o
o
o
o

1.6

4

4.4
5.5

0
2

o

o

o

2
2
0

2.0

9

4.5

0.7

4

2.8

0.8
0.9

2
2

1.6
1.8

1.0

2

2.0

6
8
11

Total I 36

17.2

55

(tnV

fV fV/onV

nR.

anR

4

0.8

o
o

0.8

o I 10

3.0
0.8

0
4
4

4.2

o

I)

o
o
o
o
o

o
o

o
o

1.2

2

2.4

1.4
1.6

11
11
8
16
25

1.7
4.6
5.5
6.7
11.4
15.6

0

o
o

o
o

o
o

24

10.6

73

6
4
2

o
o
2.5

!

o

fR fR/rmR

2.0
1.2;(2=37.67
P<O.OOI«-ratio: Mean length of each chromosome/mean length of the largest V-shaped
chromosome; nJ, nV and nR: Number of daughter chromatids in J-, V- androd-shaped chromosomes observed in this experiment; fJ, fV and fR: Frequencies of breaks in each chromosome type.

K.

GROWTH, DIFFERENTIATION AND REGENERATION
78.

Growth-Promoting Effect of Kinetin on Embryos of
Triturus pyrrhogaster Boie.
(By Yoshito OGAWA)

Kinetin (6-Furfuryl-aminopurine) was isolated first by MILLER et al.
(1956) from DNA obtained from herring sperms, and was found to be a
substance stimulating cell division in excised tobacco pith tissues. Its
effect on cell division has been reported in onion root tips (GUTTMANN,
1957), ascites tumor cells of rats (OGAWA, et al., 1957), jejunum mucosa
of mice (CUDKOWICZ, 1957) and recently, Paramecium caudatum (GUTTMANN,
1958).
This paper deals with the growth-promoting effect of Kinetin on theearly development of embryos of Trit~trus pyrrhogaster Boie. Embryos

RESEARCHES CARRIED OUT IN 1958

129

at Stage 20 were raised in Kinetin solution (10.0%0; 1.0%0; and O.l;!co) at
20 o e., and their growth with respect to organ formation was investigated.
It was found that Kinetin produced a significant effect in promoting
early embryo development, while keeping the natural balance in organ
formation. The most effective dose of Kinetin was 1 mg. per 1.000 cc.
This effect of Kinetin was first observed seven days after treatment(significant at the 1% level), and lasted for at least one week, as shownin Fig. 1. The difference between Kinetin-treated and non-treatedembryos on the seventh day was found to be four developmental stages,
which corresponds to 120 hours at 20 D e.
It is interesting to note that the time at which Kinetin becomes
effective (the seventh day in this experiment) was always Stage 30,
irrespective of the dosage and the developmental stage at which treatment was started.

1.0%0
0.1%0
Control
10.0960

Fig. 1.

Effect of Kinetin on the growth of embryos of

Triturus pyrrhogaster. Abscissa: Days after treatment.
Ordinate: Developmental stages.

79. Mitosis-Promoting Activity of a Pregnancy Serum(By Yoshito OGAWA)

The presence of mitosis-promoting substances has been recognized inearly developmental stages of animal embryos (GUSTAFSON, 1953; ANDRES.
1955; OGAWA, 1958).
The object of this note is to confirm the existence of a mitosis-stimulating substance in human pregnancy serum. The effect of pregnancy

RESEARCHES CARRIED OUT IN 1958

130

serum on cell division was investigated using Yoshida sarcoma cells
transplanted into the abdomens of Wistar rats. Normal female serum of
the same age was prepared for a control.
The pregnancy serum (ten months) was injected (2.5 cc. per 100 g. bodyweight) into the abdomens of rats (two months old) 48 hours after thetransplantation of the sarcoma, and the daily change in the frequency of
metaphasic cells was observed.
The results are given in Fig. 1.

2001
I

Injectton

Treat. 1.
Treat. 2.
Control

100

10Fig. 1. Daily change of frequency of metaphasic cells after injecting a pregnancy serum into the abdomens of rats, 10,000 cells being observed perday.
Abscissa: Days after transplantation.
Ordinate: Number of metaphasic
cells observed.

A significant increase in the frequency of moitotic figures was found in thetumor cells six or seven days after the injection of the pregancy serum(both significant at 5% level). Thus the activity of this serum in promoting cell division was confirmed. This stimulus might be due to theaction of some diffusible substance produced in the fetal tissue. The
writer has formerly pointed out that such a diffusible cell divisionpromoting substance is produced in the tissues of a regenerating liver
after partial hepatectomy.
In spite of their mitotic stimulants, somatic cells in pregnant rats wereable to keep a normal functional balance. This might not be due to theaction of any anti-mitotic substance, since the pregnancy serum showedno anti-mitotic activity in the above experiment. Whether the somatic
cells reach a stage of division or not depends upon their physiologicalcondition, as many workers have pointed out. Chemicals promoting cellRESEARCHES CARRIED OUT IN 1958

131

division, Kinetin and Na-glucuronate (OGAWA, 1958), show their activity
only in cells already preconditioned to mitosis, such as in regenerating
liver tissue after partial hepatectomy, fetal tissues or sarcomas.

80.

Synthesis of Contractile Proteins in the Early Developmentof the Embryo of Triturus pyrrhogaster.
(By Yoshito OGAWA)

The determination of the presence of actin and myosin in skeletal
muscles of the chick 72 and 96 hours after incubation, respectively, wasreported by the present writer last year. In order to test whether theappearance of actin in the developing skeletal muscle before the formation of myosin is limited to chick embryos or is a general phenomenon
in animals, the formation of skeletal muscle protein in the embryonicstages of Triturus pyrrhogaster Boie was investigated using a serological
technique. The embryo of Triturus pyrrhogaster is suitable material for
research on the biochemical mechanism of muscle protein formation,
since developmental progress is relatively slow.
G-actin and myosin were isolated from the skeletal muscle tissue of
good-sized specimens of Triturus pyrrhogaster by the method of SzentGyorgyi. Both proteins, at a concentration of 25 mg. per mI., were
injected intravenously into rabbits at three-day intervals, until theyreached a total of 100 mg. The rabbits were bled by heart puncture on
the fifth day after the last injection, and the sera obtained were raised
to their serological specificity by a resorption test with saline extracts ofliver, spleen and skin tissue of Triturus pyrrhogaster. Titration with ahomologous antigen was then carried out before the precipitin reaction
with a saline extract of the embryo. The titer of both sera was 1 : 512.
Possible failure due to an excess of antigen was prevented by using a
constant volume of serum and progressively decreasing the amount of
antigen.
It was found that actin first become detectable in the early embryo atstage 19 (about 132 hours at 20°C) and myosin at stage 24 (about 176
hours at 20°C), as shown in Table l.
Therefore, it is probably generally true that in the skeletal muscle
actin appears before the formation of myosin in the early stages ofdevelopment in animals. On the other hand, in the heart muscle of earlychick embryos, myosin appears before actin (EBERT, 1953; OGAWA, 1958).
With a view to this finding, the morphological and physiological differences of heart and skeletal muscle should be examined.

132

RESEARCHES CARRIED OUT IN 1958

The correlation between the formation of actin and myosin in early
developmental stages is now being studied.
Table 1. Determination of developing skeletal muscle proteins in early
embryos of Triturus pyrrhogaster, Boie
Developmental
stagesNo. of
Embryos
Extracted

18
19
20
23
24

12
12
12
12
12
12

25

26

--------

Control(Non-fertilized)

10No.-~f==-I...owest antigen concentrati-;;n~=~Reaction
Series
2
2
2
2
2
2
2

giving positive results*
Actin
Myosin

1
1
<1

----

24

20
40
160
1

160

< 1 : 160
< 1 : 1602* Antigen concentrations used ranged from 1 : 10 to 1 : 160 in embryo weight.

81.

Effect of Na-glucuronate on Liver Regeneration after
Partial Hepatectomy(By Yoshito OGAWA)

The writer found a promoting effect of Na-glucuronate on animal cell
division (Exp. Cell Res. 15: 415, 1958). The present report is the resultof a study to observe how the Na-glucuronate affects the regeneration ofliver tissue after partial hepatectomy. The relation between tissue weightand mitotic activity in the regenerating liver after this operation wasinvestigated using the Wistar strain of rats (two months old). The frequency of metaphasic cells was determined 48 hours after the operationin 2,000 cells per liver lobe.
The mitotic activity of regenerating liver cells after partial hepatectomy
depends upon the extent of surgical infringement. When more than 50%(by weight) of the whole liver was removed by operation, the remaining
tissue greatly increased in weight after 48 hours, but no mitotic cells
were seen. On the other hand, if the amount of liver removed was lessthan 50%, the increase in weight of the remaining part was relatively
small but the mitotic activity was quite high. Only in the latter cases
was the postoperative prognosis good (OGAWA, Y, et a1.: Med. and BioI.
45 : 12, 1957).

RESEARCHES CARRIED OUT IN 1958

133

When Na-glucuronate was injected (the most effective dose is 750 mg
per 100 g body weight), the increase in the weight of the remaining livertissue was relatively small but a remarkable increase in the mitotic index
was found in the regenerating tissue, even if more than 70% (by weight)of the whole liver was removed by the operation (Table 1).
It is concluded that Na-glucuronate may help the recovery of liverfunction and allow a good postoperative prognosis after extensive
hepatectomy which without Na-glucuronate would normally be impossible.
Table 1.

Regeneration of liver tissue after pc.rtial hepatectomy_.. - - . _ - - - - - - - - - - - - - - ---

No.
---

Sex.

Body
weight(g)- - - - " - - - - - - - ---Treated

31
32
33
34
35
36
37
38

(I)

if.
if.
if.
(I)
(I)

if.
if.

------

WeightIncrease inremovedweight oftissue regenerating livertissue (%)*
in %- - _...

147.1
159.0
106.5
125.0
120.2
98.0
132.5
169.0

37.6
45.9
52.0
62.1
67.8
71.7
79.0
88.6
- - - - - - - ..

•Control

*

1
2
3
4
5
6
7
8
9
10
11
12
13

(I)

if.
(I)

if.
if.
if.
if.
(I)
(I)

if.
(I)
(I)

if.

90.0
127.8
108.7
129.5
109.3
106.0
96.0
125.6
91.5
129.7
117.1
82.9
113.4

_--

42.8
46.4
35.5
49.6
51.0
50.5
53.6
63.2
63.4
64.6
75.0
75.0
77.3

Percent of the remaining liver tissue weight.

Mitotic
index_._---------- -

13.9
25.0
33.4
45.4
94.5
84.6
146.2
211.6
-

--- " _ . _ -

72.5
60.0
35.0
18.5
21.5
16.7
0.0
0.0

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

41.6
48.9
54.2
77.1
28.0
145.0
250.9
71.8
288.5
82.3
162.7
199.0
191.6

70.0
58.4
46.7
33.4
15.0
8.3
1.6
0.0
1.6
1.6
0.0
0.0
0.0

134

RESEARCHES CARRIED OUT IN 1958

L. TECHNICAL NOTE
82.

Simultaneous Measurement of Thermal Neutron Fluxes alld
r-Ray Dose in Thermal Column by Silver-Activated
Phosphate Glass(By Sohei KONDO)

Thermal neutron irradiation of biological materials is usually carriedout in thermal columns of reactors which are always more or less contaminated with r rays. Therefore, in such experiments it is absolutely
necessary to know the r ray dose in the presence of an intense thermal
neutron flux. The ionization chamber method so far used for thisdosimetry is very delicate and difficult. The method proposed here is avery simple and reasonably accurate way to measure simultaneously ther ray dose and the thermal neutrons. It was discovered by SCHULMAN eta1.l) that silver-activated phosphate glass exposed to X-rays acquires
the ability to fluoresce when excited by ultra-violet rays and that theamount of fluorescence is linearly proportional to the exposure dose. We
have found that the response of this glass to thermal neutrons increases
linearly with increasing dose and that the fluorescence is almost allcaused by S rays emitted from activated silver nuclei.
Experimental: The method for measuring the induced fluorescence isessentially that of SCHULMAN and ETZEL 2) except for a modification of
the holder to fit the glass plates used in this experiment. Glass platesof AgP0 3 added to a base phosphate glass (A1(P0 3)3 50%, Ba(P03)2, 25%,
KP0 3 25%) in ratios of 8:100, 10:100 and 12:100 by weight, were provided
through the courtesy of Mr. G. E. BLAIR of the Bausch and Lomb OpticalCo., Rochester, N. Y. Piles of 4 glass plates of each composition were
exposed for 24 hrs to thermal neutrons rather highly contaminated withr rays at the bottom of the water tank above the thermal column of the
Oak Ridge Graphite Reactor. 3 ) The ratio of the fluorescent output,l(x) , of a glass plate exposed in the thermal column to that, 1 (x) , of thesame glass plate exposed to 60CO r rays of 259 r is plotted in Fig. 1
against the weight percentage of AgP0 3 , x. The linear relationshipbetween l(x)jlo(x) and x, and the application to thermal column dosimetry
are explained in the following paragraph.
Theory: The fluorescent output per rad of silver-activated phosphateglass exposed to thermal neutrons is equal to that of r rayed glass.
Thus taking wD" as the energy imparted by thermal neutrons to a glass
plate of weight wand wDy as the energy released to it by r rays in the0

RESEARCHES CARRIED OUT IN 1958

135

thermal column, we may assume the following equation for fluorescent
output 1(x) :1(x)=f (x)w(Dy+D,,),(1)
where f is a proportionality constant which parametrically depends onthe concentration x of AgPO a in the glass.I) From Equation (1) wehave the equation for fluorescent output 1o(x) of the glass plate calibrated
by oOCo r rays of absorbed dose wDo
1o(x) = f (x)wDo.
(2)
From Equations (1) and (2) we obtain
1(x)/1o(x) = Dy/Do +D,,/Do.
(3)
The energy D" can be given by
Dn=(/)2jni'1ij Eu=(/)[A(l-x)+Bx]

(4)

i, j

where (/) is the number of thermal neutrons per cm 2 , ni the number of i
nuclei per gram, f1ij the cross section for any interaction (j) involving
nucleus j, Eli the total energy imparted as a result of this interaction tosecondary radiations, and x the fraction by weight of AgP03 in theglass.
Combining (4) with (3), we find1(x)/1o(x)=ax+b.
(4)This explains the linear relationship shown in Fig. 1. For the calibrazo

~

~

1~
• 1
~ ~
.5 e
~

1.5

I-

~ go

?~

e

e

e

~ 1

l eli~
e5:;
~

os

()

5
10
Weight penentnse of A~pO, in gloss (%l136

RESEARCHES CARRIED OUT IN 1958

tion exposure dose ro r of BOCO r rays, we have the following numericaF
equations:(/)=2.41·10Sroa (na"cm- Z); r=ro(b-6.05·1Q-3a) (r)(5)
Thus, from the gradient a, intercept b and calibration dose ro r, we caneasily obtain the thermal neutron flux (/) and the gamma contaminationin the thermal column, r.
From (5) and Fig. 1 we find(/)/t=6.8·10 6 nwcm-Z'see1 ; r/t=7.4 r/hr.
(6)Z
1Compared with the value (/)/t=7.1·lQfo n,,,,' cm- ·see obtained from theexposed gold foils and r/t=8.1 r/hr obtained by the ionization chamber
method"), the agreement is good. The accuracy of this method caneasily be improved by using more samples with wider differences in theirsilver concentration.
2)

J. H. Schulman et al., J. Appl. Phys. 22, 1479 (1951)
J. H. Schulman, H. W. Etzel, Science 118, 184 (1953)

3)

R. O. Bagley, et al., EPS-X-269 (unpublished) ORNL (1956)

1)

83.

The Mechanism of Serological Reactions Examined byusing Steroids(By Yoshito OGAWA)

A serological reaction obtained with cholesterol, the Wassermann
Reaction and the cephalin cholesterol flocculation test, is a useful clinical tool, although its mechanism is not well known.
The present study was made in order to determine the role of cholesterol in the cephalin cholesterol flocculation test using cholesterol
analogues. Cholesterol analogues used in this experiment were cholesteryl-acetate, cholestin-5, 7-ketocholesteryl-acetate, 3· 3-dichrol-cholesterol, and cholesten-5·diol-3·4.
In the 'case of cholesteryl-acetate, cholesten-5, 7-keto-cholesterylacetate, and 3·3-dichrol-cholesterol, negative results were obtained withall of the sera tested, but when cholesten-5·diol-3·4 was employed, reactions were positive with all of the sera except that of Kala-Azar, as.
shown in Table l.
It may be argued that a chemical constitution of the steroid, the OHradical of C3 , is an important factor in determining the results of thisreaction. It was recognized that the protein which reacts with cholesterol
remained in the serum of hepatoma patients after the resorption of theprotein which reacted with cholesten-5·diol-3·4. Furthermore, examina-

RESEARCHES CARRIED OUT IN 1958

137

tion of the serum of the same patients with cholesten-5·diol-3·4 after the
exclusion of the protein which reacts with cholesterol, gave positive
results.
These results may indicate that the protein which reacts with the OHradical of Ca is different from that which reacts with the OH-radical of
C 4 of the steroids. The results of a serological reaction using steroids
thus depend on the position of the hydroxyl radical of 1:2-cyclopentanoperhydrophenanthrein.
Table 1. Results of cephalin cholesterol floculation test using cholesterol analogues(at 24 hrs.)
-

No.
14
3
4
12
9
5
7
15
18
8
1
2
10
16
6
11
17
19

Disease

Sex

Age

-(l)-

46
40
36
32
29
38
29
36
58
20
44
27
34
34
57
19
20
41

Hepatoma+++
Necropneumonia+++
Gastroscirrhus++
Pulmonary tuberculosis++
Pulmonary tuberculosis++
Cancer of the rectum+
Pyothorax (tuberculosis)+
Cystic kidney
Cholecystitis±
Pulmonary tuberculosis±
Pulmonary tuberculosis
Pulmonary tuberculosis
Pyothorax
Purulent peritonitis
Cholecystitis
Trauma of urethra
Fracture of femur
Kala-Azar
+++

22
20
35
19
42

NormalNormalNormalNormalNormalSf'Sf'-(l)-(l)-Sf'-(l)-(l)-(l)-(l)-(l)-Sf':!;-(l)-Sf'-(l)-(l)-(l)-. - - - - - -- . _..

~ - - ~ -

101
102
103
104
105

Cholesterol(original
method)-(l)-(l)-Sf'Sf'Sf'-~------------_._--.. __ . - ---------

Cholesterol analogues
II
III
IV
V

+++
+++
+++
+++
+++
+++
+++
+++
-1-++
+++
+++
+++
+++
+++
+++
+++
+++
_-~.-._._._---.

---

+++
+++
+++
+++
+++
-------

Control- - - - - - - - - ---------- ---I. Cholesteryl-acetate.

---_... -II. Cholesten-5
III. 7-Keto-cholesteryl-acetate.
IV. 3· 3-Dichrol-cholesterol.
V. Cholesten-5· diol-3· 4.

---------RESEARCHES CARRIED OUT IN 1958

138

84.

A Simple Method for the Determination of Chromosome
Number in Rice*(By Mitsuya NEZU)

When the usual squash method is applied to the root tips of rice, goodstaining is very difficult. The writer obtained good results when theroot tips were fixed in glacial acetic acid and then squashed. For
staining, FEULGEN'S reaction was used.
Using this method, the writer determined the somatic chromosome
numbers of 2 varieties of Japanese cultivated rice and 10 strains of 8
wild species. Among them the chromosome numbers of the following 3
species were not reported before: Oryza ridleyi (WOOOl) 2n=48, O.
australiensis (W0008) 2n=24, O. alta? (W0017) 2n=48.

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

139

BOOKS AND PAPERS PUBLISHED IN 1958
BY STAFF MEMBERS•l

ENDO, T. 1958

Separation of anthocyanin and leuco-anthocyanin in flowers of
Nature 18: 801- - - 1958 Leuco.anthocyanin in variable flowers of Cam9llia japonica. Jap.
Jour. Genet. 33: 333-340
FUJII, T. 1958 Crosses between various X-ray induced recessive mutants in wheat
W.1.S. No.7: 10- - - - - 1958 Radiosensitivity in Triticum and Aegilops. W.1.S. No.7: 11--. 1958 Mutations in einkorn wheat induced by X-rays. IV. Ability of afew double recessive segregants in crosses with virido-albina to recover thechlorophyll content. Proc. Jap. Acad. 34 (9): 623-628- - - and S. MATSUMURA, 1958 Radiosensitivity in cereals. (in Jap.) Proc. II
Symp. Atomic Energy, Tokyo No.4: 185-189- - and1958 Radiosensitivity in plants. 1. Determination of LD-50
in cultivated plants (Preliminary report). Jap. Jour. Genet. 33 (12): 389-397
HIRUMI, H. and T. H. YOSIDA 1957 Sex ratio and the chromosomes of Apus
aecqualis Packard (Phyllopoda, Crustacea). (in Jap.) Jap. Jour. Genet. 32:162-164
HSIEH, S. C. and H. 1. aKA 1958 Cytological studies of sterility in hybrids between
distantly related varieties of rice, Oryza sativa L. Jap. Jour. Genet. 33 (3): 7380
!INO, T. 1958 Genetics of Phase Variation. (Abst.) VIIth Intern. Congo Microbiol.:
57-58,ISHIHARA, T. 1958 On the hyperploid cells in the regenerating rat liver. (inJap.) La Kromosomo 34-36: 1157-1161
and T. H. YOSIDA 1958 Some genetical aspects of transplantability of
the MY·mouse sarcoma. (in Jap.) Jap. Jour. Genet. 33: 23-27
IYAMA, S. 1958 Genotypic and environmental correlation in rice plants. Studies
on bulk method of breeding in plants (a book in Jap.). Yokendo, Tokyo:
146-152
KIHARA, H. 1958 Breeding of seedless fruits. (in Jap.) Seiken Ziho 9: 1-7-- 1958. 40 years after the discovery of right chromosome numbers of thegenus Triticum. W.1.S. No.7: 1-2- - - and M. TANAKA 1958 Morphological and physiological variation among
Aegilops squarrosa strains collected in Pakistan, Afghanistan and Iran. Preslia
30: 241-251- - - - K. YAMASHITA, and M. TANAKA 1958 Some aspects of the genomes of
6x species in Aegilops. Proc. Xth Inter. Congr. Genet. Vol. 2: 145
KIMURA, M. 1958 On the change of population fitness by natural selection. Heredity 12: 145-167
1958 Theoretical basis for the study of inbreeding in man. (in Jap.
with Eng. resume). Jap. Jour. Human Genet. 3: 51-70
MATSUMURA, S. 1958 Effects of temperature and irradiation time upon mutations
induced by radiations. W.LS. No.7: 5-6

Camellia japonica.

140
1958 Radiation and heredity (plant breeding). (in lap.) Proc. II Symp.
Atomic Energy, Tokyo No.4: 185-189
1958 Effects of thermal neutrons in einkorn wheat. (Abst. in lap.) lap.
lour. Genet. 33 (9): 313- - - - 1958 Effects of fl- and r-radiations in einkorn wheat. (Abst. in lap.) lap.
lour. Breed. 8 (3): 202
1958 Improvement work with sugar beets by means of triploidy. Seiken
Ziho No.9: 29-38
and T. FUJII 1958 Genetic effect of ionizing radiation in einkorn wheat.
W.I.S. No.7: 8-9
and 1958 Radiation genetics in Hordeum distichum. (in lap.)
Seiken Ziho No.9: 61-68
and S. KONDO 1958 Effect of X- and r-radiations upon wheat
seedlings and their modification due to temperature and polyploidy. W.I.S. No.
7: 9-10and M. TANAKA 1958 Use of radiation in tobacco breeding.
Proc. II Symp. Atomic Energy, Tokyo, No.4: 521-525- and S. KAWASHIMA 1958 Effect of AET upon radio-sensitivity of wheat
seeds. W.I.S. No.7: 7- , M. MURAMATSU, and S. SAKAMOTO 1958 Genome analysis in Agropyron,a genus related to Triticum. Proc. X Intern. Congo Genet., Montreal Vol. 2:
181-182
M. MURAMATSU and S. SAKAMOTO 1958 Genome analysis in Agropyron,a genus related to Triticum. Seiken Ziho No.9: 8-16
- , M. NEZU, and Y. KOSHIBA 1958 Genome analysis of Triticum georgicum.
W.I.S. No.7: 7-8, and1958 Genome analysis of Triticum georgicum.
(in Jap.) Seiken Ziho No.9: 39-48, M. SHIMOTSUMA, T. TSUCHIYA, and T. FUJII. 1958 Effects of X- and rirradiations on barley. (Abst. in lap.) lap. lour. Breed. 8 (1): 57
MORITA, T. 1958 Purine catabolism in D. mela1Wgaster. Science 128: 1135
1958. Purine contents and xanthine dehydrogenase in D. m. D.I.S. 32,
139
1958 "History of gene theory" in "Genetics and Variation" ed. by H.
Kikkawa, (in lap.), Kyoritsu, Tokyo pp. 1-16
1958 "Cytogenetics" in Ibid. pp. 17-30
NAKAJIMA, T. and H. MORISHIMA. 1958 Studies on embryo culture in plants.
II. Embryo culture of interspecific hybrids in Oryza. (in lap.) Jap. lour. Breed.
8(2): 105-110
NAKAMURA, M. and T. SUGAHARA 1958 The changes of the hardness of X-rays
transmitted by various objects and their effects on Roentgenograms. Mie
Med. Jour. 8 (1): 155-160
NAWA, S. and T. TAIRA 1958
Biochemical genetics on the eye pigmentary
system of Drosophila. (Abst. in lap.) lap. Jour. Genet. 33 (9): 326- - - - T. TAIRA, and B. SAKAGUCHI 1958 Pterine dehydrogenase found in•jBOOKS AND PAPERS WRITTEN BY STAFF MEMBERS

,
"

•

t

141

Drosophila melanogaster. Proc. lap. Acad., 34: 115-119
T. TAIRA and B. SAKAGUCHI 1958 Pterine oxidation in Drosophilamelanogaster. D. I. S., 32: 141
OGAWA, Y., Y. HARADA and T. AI 1958 Studies on the pearl oyster sex hormones I. Effects of gonade extracts on the spawing of the pearl oyster (Printada martensii). (in lap.) Medicine and Biology. 46 (1): 42-45
Ann. Report of Sizuoka Fisheries Experiment Station, Simizu 2: 11-14
and K. FUJIOKA
1958
Growth, differentiation and regeneration IV.
Observation on a Yoshida sarcoma mitotic stimulant present in rat embryo
tissue. (in lap.) Medicine and Biology 46 (3): 99-102-- -- and1958 Ditto V. Observation on a mitotic stimulant present
in pregnant sera. (in lap.) Ibid. 46 (5): 194-197-OGAWA, Y., l. MIURA and T. KAWAHARA 1958 Ditto VI. Serological determination of developing actomyosin in skeletal muscle of chick embryo. (in lap.)
Ibid. 46 (6): 242-245
1958 Studies on the mechanism of :serological reaction using cholesterol I. Cephalin cholesterol fioculation test (in lap.) Ibid. 47 (2): 54-57
1958
Mechanism of skeletal muscle protein development. (in lap.)
Kagaku 28 (6): 307
T. KAWAHARA and l. MIURA.
1958
Serological determination ofdeveloping muscle protein in chick embryo. Nature 181 (4609): 621-622
Growth, differentiation and regeneration VII. Regeneration of1958
the liver tissue of rat after partial hepatectomy. (in lap.) Medicine and Biology
48 (1): 9-12~-- 1958
Ditto. VIII. Relation between kinetin and Na-glucuronate in the
promoting effect on cell division in Yoshida sarcoma. Report on the study of
Glucuronic acid 31. Medicine and Biology 48 (3): 82-85- -- - 1958 Ditto. IX. Development of actin in the early embryo of Trituruspyrrhogaster. (in lap.) Medicine and Biology 48 (5): 201-203- 1958 Ditto. X. Effect of Na-glucuronate on regenerating liver tissue of
rat after partial hepatectomy. Report on the study of Glucuronic acid 32;
Medicine and Biology 49 (3): 96-99- - - - 1958 Mitotic activity of regenerating liver cells of rat after partial
hepatectomy. (in lap.) Medicine 15 (10): 791-794
1958 Experimental method of taste-sensitivity to bitter substances. (inlap.) The heredity 12 (12): 52-54
1958 Two chemicals promoting cell division In the Yoshida sarcoma
cells. Exp. Cell. Res. 15 (2): 415-418
1958 Development of skeletal muscle protein.
Nature 182 (4645):
1312-1313.
- - -~ 1958 Growth, differentiation and regeneration XI. Development of myosin in skeletal muscle of early chick embryo (in lap.) Medicine and Biology
49 (6): 242-244.
aKA, H. I. 1957 Genic analysis for the sterility of hybrids between distantly
related varieties of cultivated rice. lour. Genet. 55 (3): 397-409

142

BOOKS AND PAPERS WRITTEN BY STAFF MEMBERS

1958
Population-genetics studies of hybrids between distantly related
rice varieties. Studies on the bulk method of breeding in plants (a book inJap.) Yokendo, Tokyo: 27-43
~- -~
1958
Photoperiodic adaptation to latitude in rice varieties. Phyton.
11 (2): 153-160
J. HAYASHI and 1. SHIOJIRI 1958
Induced mutation of polygenes forquantitative characters in rice. Jour. Hered. 49 (1) : 11-14
and K. M. LIN 1958 Variation and selection for fertilizer response in
hybrid population of rice. (Phylogenetic differentiation of cultivated rice. 18.)
Jap. Jour. Breed. 8 (3): 163-168
OSHIMA, C. 1958 Studies on DDT-resistance in Drosophila melawgaster fromthe view point of population genetics. Jour. Hered. 49: 22-23~--~. - - 1958 The resistance of strains of Drosophila melanogaster to DDT
and Dieldrin. Amer. Nat. 86: 171-182--~-~. - - 1958 The resistance of strains of Drosophila melanogaeter to DDT and
Dieldrin. Proc. 10th Int. Congr. Genet. Vol. 2: 210- - - . - - 1958 "Population Genetics" in "Genetics and Variation" ed. by H.
Kikkawa, (in Jap.), Kyoritsu, Tokyo pp. 115-140
SAKAGUCHI, B. 1958 Enzyme protein in a strain having weak activity of tyrosinase. (Abst. in Jap.) Jour. Seric. Sci. Japan 27 (3) : 159~- and T. MORITA, 1958 On tyrosinase In insects. (Abst. in Jap.) Jap.
Jour. Genet. 33 (9): 321- - - S. NAWA and M. TSUJITA 1958
Studies on pteridine metabolism in
insects, with special reference to pteridine dehydrogenase.
(Abst. in Jap.)
Jour Seric. Sci. Jap. 27 (3) : 158~-- and M. TSUJITA
1958
On the SH radical in the yellow lethal silkworm larvae. (Abst. in Jap.) Serio Soc. Jap. Tokai branch; Kenkyu Happyokai 6:28
and M. TSUJITA 1958
Pteridine compounds in the jaundice diseasep
silkworm larvae. (Abst. in Jap.) Ibid. 6: 27
SAKAI, K. 1., R. TAKAHASHI and H. AKEMINE 1958 Studies on the Bulk Methodof Plant Breeding (A book in Jap.). Yokendo, Tokyo~-1958 Theoretical basis of breeding techniques in autogamous plants.
(in J ap.) Ibid. 3-18--~ ~ 1958
Theoretical basis of breeding techniques in autogamous plants.
(in Jap.) Ibid. 3-18
T. NARISE, Y. HIRAIZUMI and S. IYAMA 1958 Studies on competionin plants and animals. IX. Experimental studies on migration in Drosophilamelanogaster. Evolution 12 (1): 93-101
and J. J. NILES 1958 Heritability of grain shedding and other charactersin rice. Tropical Agriculturist 113: 211-218
SAKAMOTO, S. (1958)
A new early ecotype of Agropyron t.~ukushiense var. transiens. W.1.S. No.9.
SUGkHARA, T., N. KODA and T. HASHIMOTO 1958 X~ray technique in the radiological experiments. (in Jap. with Eng. resume) Annual Report of Scientific
Research Grants 1958\.

1

BOOKS AND PAPERS WRITTEN BY STAFF MEMBERS

•

r

143

TAIRA, T. and S. NAWA 1958 No direct metabolic relation between pterines
and uric acid, f1avins or folic acid in Drosophila melanogaster. Jap. Jour.
Genet. 33: 42-45
TAKAYASU, F., T. SUGAHARA and A. YANO 1958
Clinical studies on the vectorial lead of the electro-cardiogram. Mie Med. Jour. 8 (1) : 1-15
TATEOKA, T. 1958 Somatic chromosomes of Leptaspis and Streptogyna (Poaceae).
Nature 182: 1619-1620
1958 Notes on 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 Arundin13lla
and Garnotia. Bot. Gaz. 120: 101-109
1958 On the genus Streptogyna (Poaceae). (in Jap. with Eng. resume).
Jour. Jap. Bot. 33: 364-365
T AZIMA, Y. 1958 Report on sericulture industry in India. 43 pp. Central Silk
Board, India.
- - - - 1958 Changes in sensitivity of silkworm germ-cells to X-rays with
development. (Abst.) Rad. Research 9: 193- - - - 1958
Mutation response pattern of silkworm germ-cells to X-rays.
(Abst.) Proc. Xth Intern. Congr. Genet. Vol. 2: 291-292(Special lecture delivered at the1958 Radiobiology of the silkworm.
annual meeting of Tokai branch of the Sericulture Society of Japan) (in Jap.)
Proc. 6th annual meeting: 31-35--1958Inheritance of a new mutant "brown-3" of the silkworm.
(Abst. in Jap.) Jour. Seric. Sci. Jap. 27: 177- and E. INAGAKI 1958 Chromosome number of an Indian wild silkworm,
Theophila religi'osae, Westwood. (Abst. in Jap.) Jour. Seric. Sci. Jap. 27:
177
TS'UDA, F. and T. H. YOSIDA 1958 Numbers of the chromosomes and of the Vshaped chromosomes in tumor stem-cells of Yoshida sarcoma. (in Jap.) La Kromosomo, 34-36: 1190-1193
TSUJITA, M. 1958 On the Nl-U-Di complex loci. (Abst. in Jap.) Jour. Seric. Sci.
Japan 27 ,3): 180
1958 On the lethal embryos of Nh and N1 2 in silkworm. (Abst. in Jap.)
Jap. Jour. Genet. 33 (9) : 328
TSUJITA, M. 1958 Toxication of silkworm larvae by the poison emanating fromtobacco plants. (in Jap.) Kokyokigyo Chosakai, Tokyo 31-45
TSUJITA, M. S. NAWA and B. SAKAGUCHI 1958 Studies on a silkworm poison
emanating from tobacco plants. (Abst. in Jap.) Serio Soc. Japan; Tokai branch;• Kenkyu Happyokai 6: 13
and B. SAKAGUCHI 1958
Silkworm larvae with double crescent patterns obtained by X-ray irradiation. (Abst. in Jap.) Ibid. 6: 13-14
and B. SAKAGUCHI 1958
Studies on the maternal inheritance of thelethal yellow in the silkworm, Bombyx mori. II. Ovarian transplantations.
Jap. Jour. Genet. 33(7): 210-215~
1958 Heritability and genetic correlations in economic characters inchickens. Jap. Jour. Genet., 33: 13-22

144

BOOKS AND PAPERS WRITTEN BY STAFF MEMBERS

YAMADA, Y. 1958 Intended and realized selection in a White Leghorn breeding flockfor egg production (in Jap. with Eng. resume). Jap. Jour. Breed. 7: 247252, B. B. BOHREN and CRITTENDEN 1958 Genetic analysis of a White Leghorn closed flock apparently plateaued for egg production. Poul. Sci., 37:
565-580- ---, and O. KITAGAWA 1958
Polygenic mutation, induced by X-rays, inquantitative characters in Drosophila melanogaster. D.I.S. 32: 164-165
YOSIDA, T. H. 1957 Origin of V-shaped chromosomes occurring in tumor cells
and their significance for tumor growth. Proc. Intern. Genet. Symp. 1959: 210215
1957 Sex chromosomes of the tree frog, Hyla arborea japonica. Jour.
Fac. Sci. Hokkaido Univ. Ser. VI Zool. 13: 352-358
1958 Ion concentration in relation to the spreading and splitting of thechromosomes at metaphase. Proc. Symp. Cell Chern. 7: 91-100
1958 Studies on the chromosomes of Coleopteran and Hemipteran insects,
with special regard to the quantitative relation between autosomes and sex
chromosomes. Proc. Tenth Intern. Congo Entom. (1956) 2 : 979-989- - - 1958 Chromosomal alterations in malignant cells of the rat. Proc. TenthIntern. Congo Genet. (1958) Vol. 2: 328- - - - and T. ISHIHARA 1958 A karyological study of hepatoma cells in early
developmental stages of rat hepatomas. (in Jap.) La Kromosomo 37-38: 1276-1281.
I•~ftJl(~!!tlUf§'EFJf4~

·

t

~9~

8HfP 34 '¥ 9 Jj 15 8 i=[J,ffiiJ
8H'fil 34 '¥ 9 fl 20 8 961T

961T~

*

1*

OO:s'z:JI~"]tlilfY"EFJTi*J

f0*i~~ B3 JiIJ o/tF X f!~
ffil:s'z: iI~"]t ftlfY"EFJTi*J~rJJrjlU~ 6fI
1t
J* ]fit
ff: 'I'i~jT mIK 111 i!k I!lT 184*~nJrjliJFJT:a* ff: 'I'm

96 1T FJT

1E~3C~~NPIjf±

jT 1§ R: IU njz lilT 184

00 "" i! ffi ~ liJf 3t: fiJf

~IiIilIJit::='~m:&aJ

1,111

fl· (==.~) 771, 772
L -_ _

~

~

_

o
!

~.

100

200

4(]0

600
,

800

lOOOm
,