Rice Breeding with Induced Mutations II

Home , Brown rice, Maratelli, Samba (rice)

TECHNICAL REPORTS SERIES No. 102

JOINT FAO/IAEA DIVISION OF ATOMIC ENERGY IN FOOD AND AGRICULTURE

INTERNATIONAL ATOMIC ENERGY AGENCY, VIENNA, 1970

RICE BREEDING WITH INDUCED MUTATIONS II

TECHNICAL REPORTS SERIES No. 102

RICE BREEDING WITH INDUCED MUTATIONS II

REPORT OF AN FAO/IAEA RESEARCH CO-ORDINATION MEETING ON THE USE OF INDUCED MUTATIONS IN RICE BREEDING, HELD IN OISO, JAPAN, 12-14 AUGUST 1968

INTERNATIONAL ATOMIC ENERGY AGENCY VIENNA, 1970 RICE BREEDING WITH INDUCED MUTATIONS II IAEA, VIENNA, 1970 STl/DOC/10/102

Printed by the IAEA in Austria March 1970 FOREWORD

This report contains the proceedings of the fourth meeting of participants in the FAO/IAEA Co-ordinated Program of Research on the Use of Induced Mutations in Rice Breeding, a program which was initiated in 1964. The three previous meetings were reported as follows:

First: proceedings published in the International Rice Commission News- letter, Vol. XV, No. 1(1966).

Second: report presented to the IRC Working Party meeting at Lake Charles, Louisiana, 18-30 July 1966 (unpublished report, document No. 66/4468, available on request from Joint FAO/IAEA Division of Atomic Energy in Food and Agriculture, IAEA, P.O. Box 590, A-1011, Vienna).

Third: proceedings published by the IAEA as Technical Reports Series No. 86 under the title 'Rice breeding with induced mutations' .

The fourth meeting was held at Oiso, Japan, on 12-14 August 1968. Co-operators from nine countries attended, together with scientists from five other countries, the International Rice Research Institute, the Rockefeller Foundation, the International Rice Commission, and the FAO and IAEA. In addition, a number of scientists from the host country were present. The purpose of the meeting was to present reports on research related to or carried out under the co-ordinated program in 1967/68, to review and co-ordinate research plans for 1968/69, and to draw up technical recommendations for future work. With rice the staple diet of roughly half of mankind, the importance of this work, aimed at better-quality grain and higher yields, goes without saying.

CONTENTS

Mutation induction in rice by radiation combined with chemical protectants and mutagens 1 A. And o Discussion 5 Induced mutation studies with Brachiaria brizantha Stapf. and some indica rice varieties from Ceylon 7 P. Ganashan' Discussion 12 Present status of rice breeding by induced mutations in Taiwan, Republic of China 13 C.H. Hu, H.P. Wu and H.W. Li Discussion , 18 Rice breeding with induced mutations in France 21 R. Marie Mutation breeding in rice in India 25 M.S. Swaminathan, E. A. Siddiq, C.B. Singh an'd R . A. Pai Discussion 43 Haploid rice plants in mutation studies .• 45 S. T a n a ka Discussion 56

Induced mutations of rice for short-culm selections in M2 generation 57 J.H. R ee Discussion 66 Improvement of rice varieties by induced mutations to increase yield per acre and resistance to diseases and to improve seed quality , 69 A.J. Miah, I.M. Bhatti, A. Awan and G. Bari Discussion 76 Breeding for earliness, high yield and disease resistance in rice by means of induced mutations 77 M.S. H a q, S.M. Ali, A.F.M. M a n i r u z z a m a n , A. Mansur and R. Islam Induction and utilization of mutations in rice 85 G.B. Viado, I.S. Santos, E. Cada, P.B. Escuro and J.D. Soriano Induction of mutations in Thai rice varieties and subsequent selection and testing of beneficial mutant lines 105 S. Dasananda and P. Khambanonda Discussion 110 Rice stem borers in Malaya: a proposal to use mutation breeding for their control '. Ill F". C. Vohra

Recommendations 115 List of Participants 121

MUTATION INDUCTION IN RICE BY RADIATION COMBINED WITH CHEMICAL PROTECTANTS AND MUTAGENS

A. ANDO Agricultural College, University of Sao Paulo, Sao Paulo, Brazil

Abstract

MUTATION INDUCTION IN RICE BY RADIATION COMBINED WITH CHEMICAL PROTECTANTS AND MUTAGENS. Seeds of the rice variety "Dourado Precoce" were treated with different combinations of gamma rays, cysteine and EMS or gamma rays, cysteine and dES. Cysteine showed some protection against the effects of gamma radiation and combined gamma-ray + chemical treatments with regard to germination, seedling height and fertility. There are also indications of changes in the spectra of chlorophyll mutations.

INTRODUCTION

The previous report on mutation induction in rice [l] gives the results of experiments in which the seeds of the Brazilian rice variety "Dourado Precoce" were treated with different combinations of gamma rays, cysteine and ethylene oxide or gamma rays, cysteine and ethylene- imine. The highest chlorophyll mutation frequencies were obtained after the three-factor treatments with the highest radiation dose, cysteine and the respective chemical mutagen. This report deals with an experiment in which ethylmethane sulphonate (EMS) and diethyl sulphate (dES) were used as the chemical mutagens. The same rice variety was treated. Gamma irradiation was carried out in the reactor of the Atomic Energy Institute of Sao Paulo University at an approximate dose-rate of 1000 R/min, and chemical treatments were conducted immediately after irradiation at about 27°C without pH control. The concentration and treatment time of cysteine, was 0.001M for 24 h, of dES 0.03M for 3 h, and of EMS 1.5% for 8 h and 0.5% forTo h. Three panicles per plant were harvested in the sequence of flowering.

1. Gamma rays, cysteine and 1.5% EMS

Table I shows the results of the combined effects of gamma radiation, cysteine and 1.5% EMS on the rice seeds. Two-year-old seeds were used in this experiment. The protective effect of cysteine against gamma radiation, as determined by germination, seedling height and fertility, was very obvious in aged seeds. In the three-factor treatment combinations, cysteine treatment increased germination but decreased fertility as compared with the two-factor treatment combinations of gamma rays and EMS. With regard to the chlorophyll mutation frequencies, it was difficult to draw any conclusions because of the shortage of panicles in some treatment combinations.

1 TABLE I. EFFECT OF TREATING RICE SEEDS WITH DIFFERENT COMBINATIONS OF GAMMA RAYS, CYSTEINE M AND EMS (1.5% FOR 8 h)

No. of chlorophyll Seedling No. of Treatment Germination Fertility mutations height panicles per 100 panicles

O 100.0 100.0 100.0 365 1.09 OD 102.2 103.6 102.3 411 0.24 OE, 16.5 " 51.2 75.4 77 19.48 ODE, 35.2 40.9 • 33.3 50 36.00

A 102.8 82.9 71.1 374 6.42 AD 103.3 84.9' 74.1 400 6.25 AEr 8.4 32.8 19.9 13 7.69 ADE, 39.1 32.4 6.8 19 10.53

B 46.8 30.5 20.0 57 14.02 BD . 102.2 74.0 38.1 264 7.20

BE ( 0.0 - - - - BDE, 44.0 16.2 • 2.5 . 3 100.00

• C 0.0 - - - - CD 43.5 20.3 12.9 31 22.58 CEj 0.0 - - - - CDE, 1.8 8.5 0.0

O : OR A : 10 000 R B: 20 000 R C : 30 000 R D: cysteine, 1X10"3M for 24 h Ej'. EMS, 1.5°1° for 8 h TABLE II. EFFECT OF TREATING RICE SEEDS WITH DIFFERENT COMBINATIONS OF GAMMA RAYS, CYSTEINE AND EMS (0.5% FOR 10 h) OR dES (0.03M FOR 3 h) No. of chlorophyll Seedling No.of Treatment Germination ' Fertility mutations height panicles per 100 panicles

0 100.0 100.0 100.0 720 0.56 OD 97.9 100.8 100.1 1594 0.69

OE2 99.0 84.8 42.7 734 26.57

ODE2 99.6 76.7 68.0 482 24.69

OE3 97.1 83.0 96.3 732 3.28

ODE3 98.7 78.2 94.2 378 3.44

A 99.6 101.5 87-. 1 655 2.14 AD 99.4 96.4 89.3 2038 4. 02

AE2 100.2 79.8 28.6 217 19.82

ADE2 97.8 87.6 53.8 318 20.69

AE3 99.1 80.6 89.7 450 5.78

ADE3 98.1 81.2 85.3 261 8.43

B 99.2 94.7 63.0 675 6.07 BD 99.5 92.3 62.1 1606 8.22

BE2 98.8 79.7 18.5 266 22.62

BDEJ 98.2 79.6 42.4 187 20.32

BE3 98.3 68.6 46.1 556 11.33

BDE3 98.5 81.5 62.8 561 8.20

c 98.8 78.8 32.1 665 8.29 CD 96.8 ' 61.8 33.2 776 7.86

CE2 96.5 52.4 6.3 85 21.17

CDE2 100.8 57.0 19.3 252 21.82

CE3 _ 96.1 57.4 27.6 253 9.09

CDE3 ' 96.7 63.3 38.6 317 5.68

0 : 0 R B : 20 000 R D : cysteine, IX 10"3 M for 24 h E3: dES, 0 03 M for 3 h

A : 10 000 R C : 30 000 R E2: EMS, 0. 5°]o for 10 h 4 ANDO

TABLE III. CHLOROPHYLL MUTATION SPECTRA AFTER VARIOUS TREATMENT COMBINATIONS OF GAMMA RAYS, CYSTEINE AND EMS OR dES

Chlorophyll mutations Total albina viridis xantha others

Gamma rays 63 14 10 19 106

EMS 103 33 15 44 195

dES 9 5 1 9 24

Gamma rays + cysteine 160 59 8 48 275

Gamma rays + EMS 67 22 7 25 121

Gamma rays •+ dES '51 21 5 35 112

Cysteine + EMS 56 18 11 34 119 '

Gamma rays + cysteine + EMS 77 40 5 37 159

Gamma rays + cysteine •+ dES 51 10 5 20 86

631 222 67 271 1197

Xz =47.05 0.001

TABLE IV. COMPARISON OF THE CHLOROPHYLL MUTATION SPECTRA OF THE GAMMA-RAY AND GAMMA-RAY + CYSTEINE TREATMENTS IN 1967 AND 1968

Chlorophyll mutations

Treatment Total X2- P albina viridis xantha others

Gamma rays 1967 224 59 21 92 396 3.59 0.3 -0.5 1968 63 14 10 19 106

Gamma rays + cysteine 1967 292 94 22 93 501 1.70 0.5 -0.7 1968 160 59 8 48 275

1967 Gamma rays 224 59 21 92 396 4.76 0.1 -0.2 Gamma rays-t-cysteine 292 94 22 93 501

1968 Gamma rays 63 14 10 19 106 9.66 0.02-0.05 Gamma rays + cysteine 160 59 8 48 275 .ANDO 5

2. Gamma rays, cysteine and 0.5% E^VTS or 0.03M dES

The results of the different treatment combinations of gamma rays, cysteine and 0.5% EMS or 0.03M dES on the rice seeds are shown in Table II. When cysteine was applied between gamma irradiation and the application of EMS or dES the fertility generally increased compared with the two-factor treatment combination of gamma rays and EMS or dES. The three-factor treatment combinations did not cause any observ- able effects on the chlorophyll mutation frequency.

3. Chlorophyll mutation spectra

Table III shows the chlorophyll mutation spectra of various treatment combinations. This analysis was made on the data from the experiments with different combinations of gamma rays, cysteine and 0.5% EMS or 0.03M dES. Data for the cysteine and cysteine + dES treatments were excluded from the analysis because the plants had too few panicles. These results show that the chlorophyll mutation spectra are different for different treatments. A comparison of the chlorophyll mutation spectra between the treatments with gamma rays and gamma rays + cysteine was also made to see the post-treatment effect of cysteine. This comparison was based on the data from 1967 and 1968. As can be seen from Table IV, there was no significant difference in the types of chlorophyll mutation caused by the gamma-ray and gamma-ray + cysteine treatments between the two years. However, statistically significant differences between gamma-ray and gamma-ray + cysteine treatments were observed in 1968. This is particularly pronounced by the decreased frequency of xantha types of mutation in the gamma-ray + cysteine treatment compared with the gamma-ray treatment.

REFERENCE

[1] ANDO, A., in Rice Breeding With Induced Mutations, Technical Reports Series No. 86, IAEA, Vienna (1968) 7.

DISCUSSION

M.S.HAQ: Could you tell us (a) the name of the variety and (b) the moisture content of the seed during treatment? I also wonder why you got no germination after treatment with 30 000 R. A. ANDO: (a) The variety used was Dourado Precoce, one of the leading varieties in the State of Sao Paulo, (b) The moisture content was 10-12% during irradiation. In the case of two-year-old seeds we found no survivals after the 30 000 R treatment. K. MIKAELSEN: If I understood you correctly, ypu applied cysteine after the radiation exposure in your factorial treatments. Have you studied the effects when cysteine is applied before irradiation? A. ANDO: Yes, we have done a series of preliminary experiments with pre- and post-treatment of cysteine, cysteamine and glutathion. However, there were no statistically significant differences between these treatments as observed on the basis of germination and seedling height.

INDUCED MUTATION STUDIES WITH Brachiaria brizantha Stapf. AND SOME indica RICE VARIETIES FROM CEYLON

P. GANASHAN Agricultural Research Station, Maha-Illuppallama, Ceylon

Abstract

INDUCED MUTATION STUDIES WITH Brachiaria brizantha Stapf. AND SOME indica RICE VARIETIES FROM CEYLON. Non-imbibed seeds of Brachiaria brizantha were irradiated with doses of 60Co gamma rays ranging from 7. 5 to 75 kR and fresh stem cuttings containing three nodes were irradiated with doses ranging from 7.5 to.45 kR. Chromosome counts were made using the'Carn'oy method. There was no germination at 75 kR,and 18% germination at 60 kR with no survival of the seedlings. At the other doses germination and seedling survival decreased from 56% and 100% respectively for the control to 42% and 46% respectively at 45 kR. There were no chlorophyll mutants at 7.5 kR, while a. few were observed among seedlings at the higher doses. In the stem cuttings establishment was poor and morphological variations were observed at all doses above 15 kR. A mutant of desirable plant type was obtained from the 45-kR seed treatment with an erect growth habit, reduced pubescence, short internodes, profuse tillering and rapid regrowth compared with the parent. Four indica rice varieties, H-4, H-8, H-7 and Pachchaiperumal 2462/11 (PP 2462/11), were . subjected to gamma rays, neutrons and ethyl methane sulphonate (EMS) treatments. The LD50 level for gamma rays was 50 to 60 kR, for neutrons 1600 R and for EMS-0.4%. Germination and final plant stand in the M, generation were affected only slightly by the neutron- treatments, but were greatly reduced by EMS, and gamma irradiation above 35 kR. The final plant stand of the variety PP 2462/11 was most reduced by all the mutagens.

In the Mz generation there was segregation for chlorophyll mutants, of the albina, chlorina, xantha, virescens and zebrina types. Other macro-mutations observed were for grain size and shape, plant height, flowering date and sterility. Grass clump types were found occasionally..

INTRODUCTION

The use of mutagens in plant breeding in Ceylon was pioneered by Dr. S. Sakamoto (C-Plan advisor) in 1965. He treated rice seeds with 32P and irradiated them with 60Co gamma rays but did not obtain any variations from the original type. Experiments are now being conducted with rice as well as a pasture grass Brachiaria brizantha Stapf., using 60Co gamma irradiation, neutrons and ethyl methane sulphonate. Brachiaria brizantha has been found to be the-best adapted pasture grass for Ceylon's dry zone [1], Its main disadvantages are a prostrate habit, elongated internodes, pubescence, and a low seed set and viability. As the attempts to improve this grass by conventional methods of plant breeding have not been successful, mutation breeding is now being tried. The rice varieties generally grown in Ceylon have characteristically a tall growth habit, and lodge easily at high fertilizer levels. It has now

7 8 GANASHAN been shown that the highest grain yields are associated with short stiff - strawed varieties [2]. The local hybridization program for short stiff- strawed varieties is of fairly recent origin and involves only a few varieties. Meanwhile it may be possible to transform the plant type of the varieties commonly grown by using mutagens and thereby further improve their yield.

A. EXPERIMENT WITH Brachiaria brizantha

Materials and methods

Seeds and stem cuttings of Brachiaria brizantha were irradiated with the following 6(to gamma-ray doses (kR):

Seeds: . (1) 0 (2) 7.5 (3).15 (4) 30 (5) 45 (6) 60 (7) 75. Stem cuttings: (1) 0 (2) 7.5 (3) 15.0 (4) 22.5 (5) 30.0 (6) 37.5 (7) 45.0

Non-imbibed seeds were used and fresh stem cuttings of equal size, with three nodes per cutting, that were taken from a well-studied clonal selection. The cuttings were planted immediately after irradiation in a germination box. Chromosome counts were made by means of the Carnoy method.

Observations and results

The germination percentage, survival percentage of seedlings and percentage of chlorophyll mutants are given in Table I. Table II gives the survival percentage of the cuttings.

TABLE I. EFFECT OF IRRADIATION ON SEEDS

Irradiation Germination Survival percentage Percentage of treatment percentage " of seedlings chlorophyll mutants (kR)

i 0 ' 56 , 100 - 0

. 7.5 52 96 0

15.0 48 80 4

30.0 44 60 7

45.0 42 46 5

60.0 18 0 -

75.0 0 _ _ 9 GANASHAN

TABLE II. EFFECT OF IRRADIATION ON STEM CUTTINGS

Irradiation Percentage of treatment established Initial growth (kR) cuttings

0 100 Normal

7.5 75 Normal

15.0 100 Retarded growth, narrow leaves, light green streaks

22.5 75 . Retarded growth, lean leaves, reduced pubescence

30.0 50 Retarded growth, reduced pubescence

37.5 40 Severely retarded growth, reduced pubescence and increase in lethality

45.0 40 Severely retarded growth and increase in lethality

There was only a slight reduction in the number of seedlings at the lowest dose of irradiation but at the higher dosage levels seedling numbers were greatly reduced.' Chlorophyll mutants of the albina and chlorina types were observed at 15, 30 and 45 kR. Other types of morphological changes were also observed at 30 and 45 kR. Stem cuttings that were irradiated with doses above 15 kR showed variations in leaf size and growth habit together with different types of chimaeras. One mutant from the 45-kR seed treatment had.an erect growth habit with short internodes, narrow leaves and reduced pubescence.. This appeared to be the most promising mutant and was isolated for further study. Its chromosome number in pollen mother cells was 18, similar to the parent. Floral deformities and pollen sterility was also observed in this mutant.

B. EXPERIMENT WITH RICE

Materials and methods

Four indica varieties, H-4, H-8, H-7 and Pachchaiperumal 2462/11 (PP 2462/11), were treated with gamma rays, neutrons and ethyl methane sulphonate (EMS). The doses were as follows:

Mutagen Doses

I. Gamma rays (kR) (1) 0 (2) 10 (3) 20 (4) .35 (5) 50 (6) 60 II. Neutrons (R) (1) 0 (2) 300 (3) 600 (4) 900 (5) 1200 (6) 1600 III. EMS (%) (1) 0 (2) 0.2 (3) 0.4 (4) 0.6 (5) 0.8 $ 10 GANASHAN

Dry seeds were used in all treatments. Dehusked grains were used for the EMS treatment. In the Mi generation the first three panicles at flowering-were selfedby bagging and each panicle was harvested separately at maturity. The seed of each panicle was sown in a separate nursery and 25 seedlings on a line to a panicle basis were transplanted in the field at 20-21 days with a spacing of 15 cm and 30 cm between the lines.

Results

Mj generation

Treated seeds showed different degrees of germination and the final stand was affected in some by delayed lethality. The dose giving 50% lethality in each variety under different mutagenic treatments is given in Table III.

TABLE III. LD50 VALUES

Mutagens Varieties Gamma rays Neutrons EMS (kR) (R) ' (

1. H-4 . 60 1600 0.4

2. H-8 . 50 , ,1600 0.4

3. H-7 60 . 1600 ' 0.4

'4. PP 2462/11 50' 1600 0.4

Different varieties exhibited different reactions to the treatments. Chlorophyll chimaeras and stunted and deformed plants were observed mostly in the variety PP 2462/11. There was also reduction of the final ' population in H-8 and PP 2462/11.

M2 generation

In the M2 generation there were several lines segregating for chlorophyll mutants. More albina types were observed than chlorina, xantha, virescens and zebrina. Table IV gives the percentage of the M2 'segregating lines for chlorophyll mutants in the different varieties of rice. ' The most frequent mutation was for seed size with short grain types being produced more than long grain types. In H-4 there was mutation for awnedness of varying degree. Awning was rare in the H-8 mutants and the awns were usually very short. Mutants producing abnormal spikelets with, elongated sterile lemmas, large lemma and normal palea, twisted spikelets, etc. were found in H-7 and PP 2462/11. Several semi-dwarfs, • dwarfs and grass clump types were found in the taller varieties H-4 and H-8, along with a few taller type mutants. Grass 11 GANASHAN

TABLE IV. PERCENTAGE OF SEGREGATION FOR CHLOROPHYLL MUTATIONS

Mutagens ' Varieties

H-4 H-8 H-7 PP 2462/11

1. Gamma 0 (Control) 0.00 0.00 0.00 0.00 rays (kR) 10 12.51 4.28 0.00 6.25

20 11.11 10.52 1.02 4.02

35 7.31 13.46 3.07 1.02

50 9.09 15.00 1. 06 0.00

60 17.14 • - 0.00 -

2. Neutrons (R) 0 (Control) 0.00 0.00 0.00 0.00

300 2.63 9.52 3.08 4.26

600 6.79 8.73 4.68 3.22

900 10.1» 0 8.62 0.80 3.01

1200 9.59 10.09 0.00 5.62'

1600 11.82 11.26 0.00 0.00

3. EMS (%) 0 (Control) 0.00 0.00 0.00 0.00

0.2 1. 06 0.64 0.00 0.60

0.4 0.00 0.00 0.00 0.00 clump type mutants appeared only in the neutron treatments, whereas dwarfs were found mostly in gamma-irradiation treatments. Mutants with grass clump type characters did not flower and died in H-4, but in H-8 some flowered and produced seeds. There was also mutation for sterility and date of flowering. In the short aged varieties PP 2462/11 (3 months) and H-7 (3| months) there were mutants of longer age. But the medium aged varieties H-4 and H-8 (4 to 4j months) gave mutants of short and long aged types. Only changes in seed size were observed in the EMS treatment, whereas the other two physical mutagens gave a wide spectrum of mutations and several mutated characters often appeared simultaneously.

DISCUSSION

These are the first set of results obtained and they relate mainly to the morphological changes. 12 GANASHAN

In the Brachiaria mutant the erect plant habit would permit the growth of an associated legume, while the lower pubescence would improve its palatability. The rapid rate of recovery after defoliation would permit grazing at a much earlier stage of growth. In the rice varieties mutants have been selected with short stature and different dates of flowering. Estimates of herbage production in the Brachiaria mutant and grain yields of the rice mutants are still to be made.

ACKNOWLEDGEMENTS

This work was made possible with the assistance of the International Atomic Energy Agency. Grateful acknowledgement is made to the Project Leader, Dr.J.W.L. Peiris, Deputy Director (Research), Department of Agriculture, Ceylon and to Dr. G.W.E. Fernando, Agrostologist, Agri- cultural Research Station, Maha-Illuppallama who was closely associated with the investigations on Brachiaria brizantha.

REFERENCES

[1] FERNANDO, G.W. E., Grassland farming in the dry zone, Trop. Agric. Mag. Ceylon agric. Soc. 114 (1958) 183. • [2] TANAKA, A., KAWANOK, K. , YAMAGUCHI, J., Photosynthesis, respiration, and plant type of the tropical rice plant, IRRI Technical Bulletin 7 (1966) 46.

DISCUSSION

P.KHAMBANONDA: Could you tell us more about EMS treatments of dehusked grains of rice? Do you have difficulties in germinating dehusked seeds after EMS treatments? Do you think one can obtain the same mutation frequency with EMS without dehusking? P. GANASHAN: We germinated the dehusked seeds after EMS treatments under controlled hydration in petri dishes. On the third day after the seeds had been kept in controlled hydration, they were planted in seedling boxes where optimum conditions were given for proper seedling growth. The seeds receiving the highest concentration of EMS did not ... germinate and they were attacked by an ashy-brown coloured fungus. In answer to the second question, we can use husked seeds as well for EMS treatments, but I feel that dehusked seed will permit more penetration of the chemical, and the mutagenic effect will be greater. However, a detailed study is required before making firm conclusions. K.I.SAKAI: I understand that lodging is one of the most important problems in rice production in Ceylon. Did you succeed in obtaining any mutants resistant to lodging? P. GANASHAN: Lodging resistance is one of the most important objectives in our rice breeding program. From our radiation work we were very successful in isolating several lodging-resistant mutants. Lodging- resistant mutants were also obtained from the most popular variety of Ceylon, which has round grain (Samba type). Their yield potentials and •resistance to diseases have yet to be tested. PRESENT STATUS OF RICE BREEDING BY INDUCED MUTATIONS IN TAIWAN, REPUBLIC OF CHINA*

C.H. HU Taiwan Provincial Chung-Hsing University, Taichung, Taiwan and H.P. WU, H.W. LI Academia Sinica, Nankang, Taipei, Taiwan, Republic of China

Abstract

PRESENT STATUS OF RICE BREEDING BY INDUCED MUTATIONS IN TAIWAN, REPUBLIC OF CHINA. Since 1957, fourteen varieties, including both indica and japonica, have been treated with X-rays, gamma rays, thermal neutrons and EMS for inducing mutations. The objectives are: (1) To obtain erectoid mutants of good lodging resistance from the tall native varieties which can be adapted for intensive culture; (2) To obtain early maturing mutants with at least the same yield as the original variety, so that the multiple cropping system of Taiwan can be easily handled; and (3) To obtain disease-resistant mutants. The results obtained suggest that after a useful gene such as erectoid has been obtained by induced mutation, it can be used immediately. But in general, it will be more useful to combine this character into other genotypic backgrounds by cross-breeding. Henceforth, further breeding must be carried out by cross-breeding. A number of promising lines were selected from induced mutants after being crossed with local varieties and the advanced test of these lines is being carried on at present.

Since 1957, fourteen rice varieties, including indica and japonica, have been treated with X-rays, gamma rays, thermal neutrons and EMS to induce mutations. The objectives were: (1) To obtain erectoid mutants with good lodging resistance from the tall native varieties which can be adapted for intensive culture; (2) To obtain early maturing mutants with at least the same yielding ability as the original variety, so that the multiple cropping system of Taiwan can be easily handled; and (3) To obtain disease-resistant mutants. The progress reports of this breeding work have been published in various journals and the results obtained from the experiments done last year are reported in this paper.

1. Regional yielding trial of induced mutation lines in Taiwan

The X-ray-induced mutation lines of Sh 30-21 and KT 20-74 were tested in the island-wide regional tests sponsored by the District Agricultural Improvement Stations (DAIS) of Taiwan last year. The results showed that the grain yield of these two mutation lines was not

sp This is a co-operative project of Academia Sinica and Taiwan Provincial Chung-Hsing University.

13 14 SWAMINATHAN- et al.

TABLE I. RESULTS OF THE REGIONAL YIELD TRAILSa (kg/ha, 1968)

Location Taipei Hsinchu Taichung Tainan Kaohsiong Average

Variety Yield Order Yield Order Yield Order Yield Order Yield Order Yield Order

1 4116 7 4340 10 6177 7 6130 3 5055 16 5164 9 YH 1 II 3145 3 2570 4 4260 13 2905 13 3386 11 3253 7

I 3846 14 4050 15 6287 5 5270 15 5205 12 4932 16 KT 20-74 II 2715 12 2195 8 4249 14 3150 7 3100 14 3082 11

1 3959 12 4310 11 6070 9 5795 11 5075 15 5042 12 Sh 30-21 II 1590 16 2130 9 4200 16 2775 16 3040 15 2727 15

1 3908 13 4900 4 6003 10 5505 13 5181 14 5059 11 IR 9-60 II 2740 10 2080 10 4468 11 2850 14 3809 4 3189 8

4077 9 4060 14 6133 8 5365 14 5319 10 4995 14 Taichung-shen-yu 15 II 1810 14 1190 16 3850 18' 2935 10 3746 5 2706 16

I 4085 8 4960 1 6406 1 5795 11 6125 5 5474 2 Taichung-shen-yu 16 II 2968 5 1645 13 4880 5 2695 17 3410 10 3120 9

4493 2 4940 2 6306 4 6125 4 6014 6 5576 1 Taichung-shen-yu 17 II 2720 11 1765 12 4551 9 3135 9 4112 1 3257 6

3510 15 4450 9 5976 11 5805 10 5203 13 4989 15 Taichung-shen-yu 18 II 2050 13 1910 11 4229 15 2930 11 3337 13 2891 13

4238 4 4500 7 5560 17 5900 9 5997 7 5239 7 Taichung-shen-yu 19 II 2740 10 1365 14 4109 17 2820 15 3499 8 2907 12

3112 16 4240 12 6256 6 5675 12 5912 9 5039 13 Taichung-shen-yu 20 II 1703 15 1350 15 4600 8 2925 12 3705 7 3110 10

4217 5 4230 13 5936 13 6100 5 6461 2 5387 5 Kaohsiung-shen-yu 2 II 2970 4 2400 6 5228 2 3470 3 3853 3 3584 2

4085 8 4810 3 5823 14 6190 2 6355 3 5453 3 Kaohsiung-shen-yu 3 II 2878 9 3080 1 5115 4 3365 5 3364 12 3560 3

4141 6 4340 10 4823 18 6055 7 5985 8 5069 10 Kaohsiung-shen-yu 4 II 2930 7 2545 5 4701 7 3235 6 3461 9 3374 5

4276 3 4530 6 5717 16 6060 6 6534 1 5423 4 Kaohsiung-shen-yu 6 II 3473 1 . 2600 2 4497 10 3630 2 . 3925 2 3625 1

4012 10 4470 8 5740 15 6190 2 6236 4 5330 6 Kaohsiung-shen-yu 7 II 2945 6 2575 3 5292 1 3145 8 3710 6 3533 4

3964 11 4610 5 6390 2 5925 8 5225 11 5223 8 Taichung Native No. 1 II 2900 8 2390 7 3040 15 2777 14

I Taichung-shen-yu 7 ' II 4313 12

I Taichung-shen-yu 14 II 4701 6

I Taichung-shen 2 II 3370 4

I Ai-chio-chien II 3435 2

I Min-Tang-Tsieh II 5168 3

I Chung-Lin-Chung II 4150 1

3 Data from Department of Agriculture and Forestry, Taiwan Provincial Government, April 1968. HU et al. 15 higher than the other short-stature varieties which were offered by the DAIS. YH 1, a selected line from the hybrid of T(N) 1 X Sh 30-21, yielded more than its parents. The YH 1 yield exceeded that of T(N) 1 by about 3.5% in Taipei and Tainan in the first crop and by 17% in Taipei, Hsinchu and Kaoshung in the second crop. .But YH 1 was not the highest yielding variety in this trial (see Table I). These regional trials will be continued.

2. Extension of YH 1 and YH 2 trials by farmers in Chaiyi and Luckon

Before a new variety is recognized by the Government it must pass the above regional yielding trial for at least two years. The demonstration of new varieties together with local ones will be taken up by the DAIS and/or by the Farmers' Association of Taiwan (FAT). Although YH 1 is still on trial it has proven to be very successful in the Chaiyi area near the tropic of Cancer where it was grown on about one hectare of farmland. The data show that YH 1 yielded 7050 kg/ha in the second crop of 1967 and 8487 kg/ha in the first crop of 1968. These two crops out-yielded T(N) 1 (the check) by 30% and 19.4% respectively (see Table II). Because of the excellent results obtained, FAT in Chaiyi is going to extend YH 1 to about 50 hectares in the second crop of 1968. YH 2 and YH 3, the selected lines from hybrids of T(N) 1 X KT 20-74 and T(N) 1 X Sh 30-21 respectively, were also grown on a small scale in Chaiyi. As shown in Table II the grain yield of YH 2 is almost the same as that of YH 1, whereas YH 3 yields less. But YH 3 is an early- maturing variety that 'matures 7-10 days before T(N) 1. YH 2 also grew well in Luckon, a coastal region near Taichung. This is a monsoon region with strong seasonal winds in the late fall, so the rice yield never reaches 6000 kg/ha per crop. YH 2 was able to reach the goal with a yield as high as 6094 kg/ha in the first crop in 1967. The Farmers' Association in Luckon is also planning to extend YH 1 and YH 2 in 1969.

TABLE II. RESULTS OF THE DEMONSTRATED TRIALS IN CHIAYI REGION a (kg/ha dry seeds)

Variety T (N) 1 YH 1 YH 2 YH 3 Duplication (check)

I 8208 8504 8056 6948

II 8568 8680 7996 7452

III 8280 8120 7292 7488

IV , 8892 8504 8668 6552

Average 8487 b 8452 b 8003 b 7110

% 119.6 118.9 112.6 100.0

a Data from Farmers' Association in Chiayi. k Significant at lfo level. M.R.T.: Dd) = 680, 15(3 ) = 660, 0(^ = 628. 16 SWAMINATHAN- et al.

3. Cross-breeding in Taichung

Taichung is the agricultural centre of Taiwan. The rice varieties from Taichung, e.g. Taichung 65, T(N) 1, have been developed by cross- breeding a variety with a good performance and one with wide adaptability. Sh 30-21 and KT 20-74 were first developed in Taichung and are now used as parents for cross-breeding. The following hybrid lines are being tested in the experimental field of Chung-Hsing University.

/ F2 generation. 17 cross combinations were obtained with the particular aim to obtain resistance to blight disease. The resistant parent varieties were kindly supplied by Dr. T.T. Chang of IRRI, Philippines.

F3 generation. 182 lines were selected from the annual F2 populations of KT 20-74 X IR-8 and others. According to reports of IRRI and Chang [1, 2], IR-8 responds well to nitrogen fertilizer, has strong culms and wide adaptability in tropical countries. But IR-8 is too late and low yielding in sub-tropical zones such as Taichung, and is not cold resistant at the nursery stage. However, the F2 population shows a wide range of variation which enables selection of some early types with good tillering ability and resistance to leaf stripe disease.

F6-7 generation. 11 of 29 lines exceeded T(N) 1 in yield in the first crop of 1968. This trial will be continued in the second crop of 1968. Hence the grain yield of T(N) 1 is good for the first cropping season in Taiwan but not so good in the second cropping season. High yielding strains, particularly in the second cropping season, are being sought.

F3-9 generation. The advanced yielding trials were carried out in four places around Taichung. The results showed that the yield of 641-5121 exceeded T(N) 1 by 10.4 and 5.3% in Erh-Fen Pu and Erh-Lin respectively, but was less in the experimental fields of Chung-Hsing University. On the other hand the YH 2 yield was better in Luckon but less in the above two places. The early-maturing variety, YH 3, gave poor yields in these trials.

DISCUSSION

At the beginning of the radiation breeding work, it was believed that radiation might be useful to improve the weak characters of the existing rice varieties in one stroke, such as lodging resistance, disease resistance and early maturation, etc. As a result, the induced mutants with similar genotypes were considered to have the characters of the parent varieties, except the mutated characters. Also, the method seemed to be more attractive and effective than conventional breeding. The successful use of radiations by Gustafsson to induce favourable mutants in barley and by Gregory to induce resistance to leaf spot disease in peanuts gave us courage to enter this work. But we did not take into account the fact that the varieties used by the farmers

1 A selected line from the hybrid of Siam's King X Sh 30-21. HU et al. 17

TABLE III. YIELD OF indica ERECTOIDS IN THE REGIONAL TRIALS IN OUR PROGRAM (1st. crop, 1968)'

Location Chung-Hsing Luc kon Erh-Fen-Pu Erh-Lin Average University Variety •

Taichung kg/ha 5476 6670 7053 7580 6694.8 Native No. 1 (as check) % . 100.0 100.0 100.0 100.0 100.0

YH 2 % 101.4 103.0 99.4 97.2 100.4 63 11-21123

YH 3 % 94.0 86.5 97.8 79.5 89.5 63 II-1441

63 11-1642 1° 93.1 ' 94.6 97.5 90.1 93.8

64 1-512 % 92.9 100.8 110.4a 105.3 102.4

As-60 to 89.7 94.2' 100.2 90,8 93.7

64 1-541 % 89.4 90.0 93.5 - 95.3 92.1

As-AK % 86.5 88.0 95.9 94.3 91.2

63 11-1551 % 85.4 86.5 95.5 82.2 87.4

64 1-111 % 84.9 91.7 105.4 93.8 94.0

64 1-311 °lo 84.2 98.5 101.6 95.3 94.9

63 11-3672 % 81.8 . 91.5 99.7 . 90.2 90.8

a Significant at 5% level. were also changeable. In Taiwan, because of the great improvement in agriculture and the farmers' knowledge, the native varieties have now completely changed.- When we started the mutation breeding work, almost all the native varieties were tall, lodging and not intensive. We are seeking erectoid mutants of different varieties and frequently em- phasize this point. The short-stature variety, such as T(N) 1, and others were gradually but extensively taking over. The trait of lodging resistance is no longer sufficient for the farmers' needs. The intensive, early, disease resistant and high-yielding varieties are required. As a matter of fact, the farmers have put a lot of effort into seeking any new, good variety even without guarantees from the Government. The present situation is that we should study any possible method of breeding. As previous papers have indicated [3,4] the semi-dwarf gene of induced erectoid mutants from different varieties, was considered to be at the same locus as that being found from spontaneous origins (Dee-geo- woo-gen, a parent of T(N) 1), but different yielding according to the 18 SWAMINATHAN- et al. genotype of other characters interacting with the same semi-dwarf gene. The component characters of grain yield, therefore, would be different. As a matter of fact the varieties in Table I are all the same type as T(N) 1, but with different grain yields. The induced erectoid mutant would be a good example for further trials in induced mutation breeding in the future, not only for direct field use but also for indirect use as parental material for cross-breeding.

SUMMARY

1. The induced mutants of Sh 30-21 and KT 20-74 were tried out in the Taiwan island-wide regional trials in 1967 and showed that the yield was a little less than that of the variety YH 1, a selected line from the hybrid of T(N) 1 X Sh 30-21. The yield from YH 1 exceeded T(N) 1 by 3.5% in Taipei and Tainan in the first crop and by 17% in Taipei, Hsinchu and Kaoshung in the second crop. But YH 1 was not the highest yielding variety in this trial. 2. YH 1 is particularly good in the Chaiyi area (between Taichung and Tainan). A comparison of the yields of YH 1 and T(N) 1 in this area by large-scale demonstration trials showed that the grain yield of the former was 7050 kg/ha in the second crop of 1967 and 8487 kg/ha in the first crop of 1968, which showed that the former out-yielded the latter by 30% and 19.4% respectively. The Farmers' Association of Taiwan in Chaiyi city is going to extend YH 1 to 50 hectares in the second crop of 1968. 3. ' YH 2, a selected line from the hybrid of T(N) 1 X KT 20-74, has a similar grain'yield to YH 1 and proved to be particularly good in Luckon, a coastal area near Taichung. The Farmers' Association of Taiwan in Luckon also plans to extend YH 2 in 1969. 4. Cross-breeding was carried out by using induced mutants of Sh 30-21 and KT 20-74 and other good performance varieties. The generations F2 to F1() are now being tested.

REFERENCES

[1] The International Rice Research Institute Ann. Rep. 1966, (1967) 65-66. [2] CHANG, T. T., The genetic basis of wide adaptability and yielding ability of rice varieties in the Tropics, Int. Rice Commn Newsl. 1£(4) (1967) 4. [3] LI, H.W.. HU, C.H., WOO, S.C., Further report on mutation breeding of rice in Taiwan since 1957, Bot. Bull. Acad, sin., Shanghai 6 (2) (1965) 131. [4] LI, H.W.. HU, C.H., WOO, S.C., Induced mutation breeding of rice in Taiwan, Ibid. 7(2) (1966) 71.

DISCUSSION

K.I. SAKAI: You mentioned that several semi-dwarf mutants induced by irradiation were the same as those resulting from spontaneous mutations. Is there any variation among those mutants with respect to dwarf- ness or other characters ? Are these mutant genes perhaps multiple alleles on the same locus?. HU et al. 19

C.H. HU: One does find different characters among the semi-dwarf mutants obtained from different varieties. Diallel crosses have been made between T(N) 1 and induced semi-dwarf mutants, but no segregation was found with regard to height. In the cross KT 20-74 X IR-8, about 0.2% of the F2 population consisted of tall plants. This suggests that different sites of the semi-dwarf gene may be present. M.S. HAQ: Are the mutant varieties which lost short-day sensitivity, also insensitive to temperature? C.H. HU: There was no change with regard to temperature sensitivity.

RICE BREEDING WITH INDUCED MUTATIONS IN FRANCE

R. MARIE Station d'Amelioration des Plantes, Institut National de Recherches Agronomiques, Montpellier, France

Abstract

RICE BREEDING WITH INDUCED MUTATIONS IN FRANCE. Mutation experiments with rice at . Montpellier yielded strains with improved lodging resistance, grain size, maturing time, milling quality and other characters. The general performance of these mutant strains was tested in field trials. Further mutagenic treatments were made to improve the high-yielding short grain varieties with regard to grain quality and seed dormancy.

At the Plant Breeding Station of Montpellier (South of France), studies have been conducted on rice mutation since 1957. The number of varieties able to ripen in the French Mediterranean region is relatively low compared with the number of rice varieties in the world, chiefly because of the photoperiodic response: most foreign varieties, being adapted to shorter day-lengths, cannot flower in our rice area (43° 25' to 43° 50' latitude N), and many of the varieties which can flower do not ripen. In 1968 our collection contained 130 varieties from 18 countries; these are the only varieties retained for further study after testing 2335 strains, originating from 48 countries of various lati- tudes, during 21 years. Some of the objectives in our rice improvement program, i.e. yield,^ yield regularity and quality of the grain, have been reached by normal crossing and breeding methods. The first desired change by mutation was to produce a good resistance to lodging in the early varieties, which •• were all susceptible under our environmental conditions (Allorio, Maratelli, Precoce Corbetta, Bellardone, Ishikari-Shiroke). We began in 1957 to irradiate Allorio, Maratelli and Precoce Corbetta .with gamma rays. Our. most spectacular mutant, Allorio "Lambda" (named " Professor Lamarque"1) has a shorter culm, a better resistance to lodging and an increased yield (about 50%) in comparison with the starting variety Allorio 11. On the other hand, it ripens about 6 days later and its milling quality is in- sufficient: this interesting mutant encouraged us to continue with mutation. Maratelli gave only a chlorophyll mutant and Precoce Corbetta two erectoid mutant lines with a bigger grain, but not a superior yield. The mutation program was extended to other mutagens (such as X-rays and EMS) and to other varieties: Cesariot, Stirpe 136 X Sesia, Americano 1600, Balilla 28 and Cigalon. Besides useful variations, which are a very small minority of the induced mutations, we detected in the progenies of treated seeds a great number of types of botanical interest, but without practical value for rice growers.

1 Professor P. Lamarque, head of the Centre de Lutte contre le Cancer, Montpellier, has given us valuable help since 1957 with his assistant Dr. J. Garv-Bobo.

21 FAO/IAEA/IRRI UNIFORM YIELD TRIAL FOR IRRADIATED japonica RICE MUTANTS AND HYBRID SELECTIONS

RESULTS IN FRANCE • ,

A. GENERAL DATA

Harvest year: 1967

Location; No. 11

Location name: Saujan (Gard) France

Annex of Montpellier Plant Breeding Station.

Worker's name: R. Marie

Seeding date: 6 May, 126th day of the year •

Harvest date; 18 October, 291st day of the'year

Experimental design; 4 randomized complete blocks

Plot area: 5.58 m1 (1.20 X4.65)

Number of seedlings per hill: 1 (2 grains sowed, then adjusted)*

Row distance: 30 cm

Hill distance: 15 cm

Latitude: 43 degrees 44 minutes (N)

Longitude: 4 degrees 37 minutes (E)

Precipitations during the test period: 68.6 mm - Irrigation: about 2000 mm

Fertilizer applied to the experiment: NPK: 100, 144, 72

form: (NH4)jS04; Ca(P04)2 ; K,S04

dates: for P and K: 12 April; for N: 27 April and in top dressing 17 June

application; by hand

Seasonal climatic conditions: very good.

No transplanting because of the lack of water for a nursery. B. RESULTS

1. General behaviour

Average Date of Maturing characteristics Variety Country height Remarks on 18 Oct. (normal date of harvesting) heading (cm)

Normal (i) Allorio 11 , Control France 1 Aug. 135 Harvested and tested

(2) Allorio Lambda Mutant from (1) • France 2 Aug. 90 Harvested and tested

(3) Balilla Local check France 14 Aug. 85 Harvested and tested

Late (4) R. 90 Mutant from (9) Japan 18 Aug. 95 Harvested and tested

Uncertain (5) Fukei 71 Mutant from (7) Japan 18 Aug. 65 Spikelet sterility: 20 to 30%. Harvested for a new trial in 1968

(6) Reimei Mutant from (7) Japan 19 Aug. 85 Irregular germination and growth. Harvested for a new trial in 1968

(7) Fujiminori Control Japan 17 Aug. 95 Irregular germination and growth. 90% unripe grains. Harvested for 1968

(8) San In 73 Mutant from.(12) Japan 20 Aug. 95 Irregular germination and growth. Harvested for a new trial in 1968

Not maturing 0) Koshihikari ' Control . Japan 10 Sept. 95 Hard dough stage

(10) From Taichung 65 Mutant Taiwan 11 Sept. 90 Soft to hard dough stage

(11) Tainan 3 Hybrid Taiwan 12 sept. 95 Soft dough stage

(12) Notin 22- Control Japan - 20 Sept. 100 Complete heading but spikelet sterility 100%

(13) FF 36 Hybrid Taiwan 2 Oct. 70 Half-headed

2. Other data on rice varieties

Average yield Panicles/m* (4 blocks) (av.) Panicles/plant (av.) ' Yield g/plot (14.5 % grain moisture) (av.) (g/ha)

Allorio 11 206 175 211 200 - 198 9 3 7.9 9.5 9 0 8.9 2305 1705 1970 2225 2051 39.3 Allorio Lambda 368 313 301 353 334 16 6 --14.1 13.6 15 9 15.0 . 3705 4100 3660 3300 3691 70.7 Balilla 304 278 264 311 .', 290 13 7 • 12.5 • 11.9 14 0 - 13.0 - 4285' 3495 3705 3550 3759 72.0 R. 90 422 412 420 387 411 19 0 18.6 18.9 17 4 18.5 3925 4395 3670 4000 3998 76.6

L. •d. (0.05) 10.0 24 MARIE

At the present time our Station possesses 195 mutant strains screened in these mother varieties and selected according to the following two criteria:

1. For characters of agricultural interest:

With X-rays: 4 mutants less sensitive to lodging, with larger grain, and earlier maturity. With gamma rays: 84 mutants with shorter and stronger culms, more resistance to lodging, increased yield, earlier and later maturity, larger and slender grain, better milling quality, or better commercial quality. With ethyl methane sulphonate: 7 mutants with shorter and stronger culms, more resistance to lodging, increased yield, earlier and later maturity, larger and slender grain, better milling quality, or better commercial value.

2. For characters without practical value;

100 mutant lines, obtained after treatments with X-rays, gamma rays and ethyl methane sulphonate. They are kept in the stock collection only for teaching purposes and theoretical studies.

Our first good mutant, Allorio Lambda was discarded in 1965 for its poor milling quality. On the other hand, in 1968 we began to test the performance of a larger grain mutant, induced in Cesariot in 1961 by gamma rays, which satisfies the present-day requirements of the French market. The short grain varieties, which are much appreciated by the rice growers for their high yield, need to be improved in respect of their commercial value: they should be entirely vitreous or translucent and without chalky spot (white belly). We have worked towards this goal since 1963 and some strains without spot have been induced in the varieties Balilla and Cigalon. They are still under selection. We must point out that the "vitreous" mutants present, in addition, some changes in the grain size and shape and in the duration of the vegetative cycle. Finally, we are carrying on a mutation program for inducing dormancy in the non-dormant variety Cigalon: this type of mutation is very easy to screen and interesting for a physiological study of dormancy in seeds. MUTATION BREEDING IN RICE IN INDIA

M.S. SWAMINATHAN, E. A. SIDDIQ, C.B. SINGH, R.A. PAI Indian Agricultural Research Institute, New Delhi, India

Abstract

MUTATION BREEDING IN RICE IN INDIA. Mutation research was continued in rice with the following aims: (a) to enhance the frequency and spectrum of mutations in indica and japonica rice varieties; (b) to change the grain quality of'the japonica variety, Tainan-3f into the indica type; (c) to improve the grain quality of the indica variety, IR-8; (d) to increase the recombination frequency in japonica-indica hybrids. Both nitrosoguanidine and 5-MeV fast neutrons gave a high mutation frequency. The japonica variety was more sensitive to all mutagens than the indica types. Chemical mutagens had no particular advantage over ionizing radiations with reference to either mutation frequency or spectrum. Mutants with indica type of grain occurred readily in Tainan-3 in all treatments. Such mutants had a larger grain length/ width ratio and were more resistant to alkali digestion.

Fine grain types with better cooking quality occurred in.the M2 populations of IR-8. These mutants are likely to render this high-yielding variety more popular. A wide range of chlorophyll and viable mutations occurred in IR-8 and Tainan-3. Some of these, like those involving dwarfing and slow senescence, are of economic interest, besides those affecting grain quality. Recombination frequency can be influenced in japonica X indica hybrids through the irradiation of Fj sporocytes. The precise influence varies with the stage at which the plant is irradiated, the dose given and the loci involved.

INTRODUCTION

The development of high-yielding semi-dwarf indica varieties in Taiwan and at the International Rice Research Institute, Philippines, by using the Dee-geo-woo-gen dwarfing gene has helped to dispel the old notion that japonica (ponlai) varieties alone are capable of responding well to the application of fertilizers. Dwarf indicas such as T(N)1 and IR-8, and several tropical japonicas like Tainan-3, Taichung-65 and Kaoshiung-68 have done well in India and have yielded from 5 to 9 tons per hectare. Despite their outstanding features like high-yielding po- tentiality, photo-insensitivity and synchronized tillering T(N)1 and IR-8 are becoming unpopular because of their coarse grain, inferior cooking quality and high susceptibility to bacterial leaf blight and tungro virus. Although the ponlai varieties like Tainan-3 possess a high degree of resistance to bacterial blight and can give high yields,' they are unpopular in India because their grains become sticky on cooking. Since the release of IR-8 another culture, IR-5, which possesses a high head milling rice recovery with a relatively higher degree of resistance to various diseases and better cooking quality than IR-8, has been evolved at the IRRI. Extensive studies on IR-5 in India have shown that it matures very late. Keeip'ing in mind all the merits and demerits of these recent introductions, mutation breeding was initiated with the following objectives:

25 26 SWAMINATHAN- et al.

(a) Mutational rectification of grain size and cooling quality in IR-8 (b) Conversion of the grain quality of ponlai types (Tainan-3) to that of the indica type (c) Induction of mutations for earliness in IR-5.

Besides taking up work on these applied aims, systematic studies were carried out on the maximisation of mutation frequency and spectrum in indica and japonica rices. The non-recovery of desirable recombinations, which is presumably due to either cryptic structural differences or other genetical factors resulting in tight linkages, and also defective screening procedures appear to be the main reasons for the failure of the indica X japonica hybridi- sation program sponsored by FAO in 1951. Since radiation can be used as a tool for increasing recombination, studies were undertaken to measure recombination frequency in F2 populations derived from irradiated Fj material.

MATERIAL AND METHODS

In studies on the maximisation of mutation frequency and mutational rectification of undesirable traits, three high-yielding varieties (two representing indica and one japonica subspecies of (X sativa) were used. The salient features of these varieties are given in Table I. For studies on the influence of radiation and chemical agents on the recombination frequency in japonica X indica hybrids, the following material, kindly supplied by Dr. T.T. Chang, was used. Parental combination Marker genes involved

1. N-45X IR-8 pusur (Japan) N-45 : d6 .-Top leafed dwarf CBP- Chromogen A -Activator PI - Purple leaf 2. 1-45 CharnackX A-58, Kokushopento-2 ' A-58 Kokus (Japan) . CB - Chromogen A -Activator Pr - Purple hull Ph - Phenol staining Pn - Purple node , Wx - Waxy glutinous

Similar studies were also taken up in reciprocal combinations of the following crosses which, however, do not have distinct markers:

(a) IR-8 Tainan-3 (b) T(N)1 X Tainan-3 (c) Basmati 370 X Tainan-3

Method

Seeds with 12 - 13% moisture content were treated with different doses of gamma rays, fast neutrons, ethyl methane sulphonate (EMS), SWAMINATH AN et al. 27 nitrosomethylurea (NMU) and nitrosoguanidine (NG) at a constant temperature of 28°C. In the chemical treatments, seeds pre-soaked for 5 h were immersed in the aqueous solutions of the mutagen with intermittent shaking for 8 h at pH 7. The M2 generation was grown from the seeds of selfed M1 plants. The M^ plant progenies and the M-^ spike progenies were kept separate.

RESULTS AND DISCUSSION

I. Mutation studies on indica and japonica varieties

A detailed study of the effects of physical (gamma rays and 5-MeV fast neutrons) and chemical (EMS, NMU and NG) mutagens was carried out on IR-8 (indica) and Tainan-3 (japonica). To assess the relative efficiency and effectiveness of nitrosoguanidine, which is a new addition to the series of potent chemical mutagens, in comparison to that of gamma rays, EMS and NMU, a detailed study was made on IR-8. Data on the percentage of Mi survival indicated that the toxicity of NG and NMU -was very high, followed by that of gamma rays and EMS. The LDS0 seemed to be 30 kR with gamma rays and 0.5%, 0.015% and 0.010% with EMS, NMU and NG respectively (Table II). Data on the frequency of chlorophyll mutations, as measured by the percentage of M2 families segregating, indicated that NG induced the most and NMU the.least at biologically equivalent doses. Although there was a positive correlation between lethality and mutation frequency in gamma-ray, EMS and NG treatments, it was quite the reverse in treatments with NMU (Table III). The order of mutagenic efficiency and effectiveness, as measured by the scheme suggested by Konzak et al. [1], of the mutagens used were as follows (cf. Table IV):

NG > NMU > EMS > gamma (Effectiveness)

NG > EMS > gamma > NMU (Efficiency)

Another characteristic of the NG treatments was the greater recovery of mutations, as expressed by a better fit to the expected segregation ratios (Table V).. Chemicals, compared with physical mutagens, tend to yield a lower segregation ratio, probably due to the operation of a less rigorous somatic sieve (cf. Ref.[2]). The percentage of mutations in segregating .families in NG treatments was about 23 as against about 17 and 19 in gamma-ray and EMS treatments respectively (Table V). This is probably due to a lower number of sector cells partaking in the formation of an Mj spike. . Thus, both for induction and recovery of mutations, NG seems to be quite valuable for use in rice.

Studies on fast-neutron treated material

Seeds of IR-8 and Tainan-3 were treated with 5-MeV fast neutrons ' through the help of the IAEA, Vienna. The percentage of survival showed almost a reverse trend beyond the dose level of 2400 rads in IR-8 and 1800 rads in Tainan-3 (Table VI). 28 SWAMINATHAN- et al.

The frequency of chlorophyll mutations in the M2 generation, as measured by the percentage of Mj spike progenies segregating, was 45. 2% in Tainan-3 and 28. 6% in IR-8. However, as in the case of survival, the dose-reversal phenomenon also occurred with regard to mutation frequency (Table VII). This could be due to the onset of a more rigorous diplontic selection at doses higher than the optimum level. Another interesting feature about the neutron treatment was its ability to induce a wider spectrum of chlorophyll mutations than other ionizing radiations and chemical mutagens (Table VIII). The data show that the frequency of albina was highest compared with the other types and that it was dose dependent. This may be due to the larger number of loci governing this phenotype. Varietal variation was evident from the relative frequencies of chlorophyll mutation types as shown in Table VIII. Mutagenic potency indicated that the effectiveness was uniformly highest at lower doses and that the values seemed to be 7-10 times more than with gamma-rays and 2-3 times more than with EMS. Efficiency and dose were inversely correlated (Table IX). Studies on the effects of neutrons on the spike initials as measured by the segregation ratio for albina seedlings indicated that the sector size was influenced by varietal difference. For instance, the ratio was nearer the expected value (25%) in IR-8, whereas the ratio in Tainan-3 • was half the value expected (Table X). The frequency of chlorophyll and viable mutations in Tainan-3 and IR-8 after treatments with different doses of gamma-rays, EMS and NMU (Tables XI and XII) suggests that the frequency of chlorophyll mutations in Tainan-3 after gamma-ray and EMS treatments was more or less the same, whereas it was considerably lower after the NMU treatments. The trend in viable mutations was almost the same as in chlorophyll mutation. Unlike with Tainan-3, IR-8 showed the highest mutation frequency after gamma-ray treatments followed by NMU and then EMS. Although the chlorophyll mutation frequency after EMS treatments was the lowest, viable mutation frequency after these treatments was on par with that of gamma-ray treatment. NMU induced a relatively lower mutation frequency (both viable and lethal) compared with gamma-ray treatments. These findings are in agreement with our earlier results with rice that gamma-rays are as potent as any efficient chemical mutagen known.

Mutants of economic interest

The isolation of indica-like grain mutants in Tainan-3 and of fine grain types in IR-8 being the 'applied' objectives, the M2 population was carefully screened for grain characters. It is evident from the data in Table XIII that mutations for indica grain in gamma-ray treatments are as high as in EMS treatments. It is also apparent that the frequency of their occurrence has a tendency to be higher when dehusked seeds were treated. Alkali digestion tests of these mutants revealed that they are superior to their parents in having a moderate gelatinization temperature. Apart from their economic value, the occurrence of such mutations lends further support to the view that the sub-specific differentiation in O. sativa has proceeded by the clustering of mutations probably under the influence of disruptive selection. The occurrence of fine grain mutants in IR-8, as judged by the increased L/B ratio, suggests that SWAMINATH AN et al. 29 mutation breeding can be a potent tool for altering the grain characters. The KOH test (2.4%) at 28°C for 24 h revealed that the fine-grain types withstand disintegration better than IR-8. Seeds are being multiplied for large-scale yield trials during 1968. A list of the major types of viable mutations recorded in IR-8 and Tainan-3 is given in Table XIV.

II. Enhancement of mutation frequency by synchronising the time of mutagen treatment with the DNA synthesis

Dehusked seeds of Tainan-3 and Taichung-65 were treated with 1% EMS for one hour after pre-soaking periods of 8 to 22 h (Tables XV and XVI). The data on the percentage of germination and survival show a drastic reduction in the seed lots presoaked for a period ranging between 18 and 22 h. The mutation frequency and spectrum in these treatments are being studied.

III. Increasing the diffusion rate of chemical mutagens

Dimethyl sulphoxide (dMSO) is considered to be an efficient carrier of chemical mutagens in higher plants. Earlier reports by various workers indicated that dMSO has no apparent inhibiting effect on the growth of the treated plants. The present findings in rice, however, show a marked effect of the chemical on germination and survival (Table XVI). The percentage of seedling lethality after dMSO treatment was as high as after EMS and dES treatments. Combined treatments of EMS (l%) + dMSO (5%) also showed the same levels of germination and survival as with dMSO (5%) alone. The effects of dMSO in combination with EMS on mutation frequency is being assessed.

IV. Promotion of crossing-over in indica X japonica hybrids

Studies were carried out on: (a) The effects of radiation and chemical treatments on chiasma frequency and panicle fertility; and (b) The recombination frequency in the F2 progenies derived from normal and irradiated F1 plants.

Studies on the immediate effects of the mutagenic treatments as measured by the chiasma frequency (Table XVII) revealed that there was a significant increase after radiation treatments. In general, the dose and the number of chiasmata were found to be inversely correlated, except for the doses 240 and 480 R (Table XVII). To study whether the sensitivity is different at different stages of flower development and whether there are significant differences caused by a lapse of time between treatment and fixation, panicles were fixed at two different intervals, i.e. 24 and 48 h. The data show a decline in the chiasma frequency with an increased period between treatment and fixation. The average panicle fertilities in the control as well as in treatments with X-rays and mitomycin C (MC) were nearly the same. 30 SWAMINATHAN- et al.

Recombination frequency

The recombination studies in the cross N-45X 1-68 (pusur) restricted to two genes, namely d6 and PI, revealed that the recombination percentage was considerably increased after X-ray and MC treatments given at the pre-meiotic stages (Table XVIII). Treatments at the post-meiotic stage showed the same or a lower recombination percentage than the control. In the non-treated branches of the same plant there was a drop in the percentage of recombination, except at 240 R (Table XIX). Similarly, in the second cross between I-45(Charnack)X A-58 (Kokushopento-2) recombinations for various markers located within and between linkage groups were calculated in the control and treated populations (Table XIX). It was observed that the response to radiation of the recombination frequency for different gene combinations differed significantly. The influence of mutagens on the recombination of genes within the linkage group was found to show an increase for genes CB and WX, whereas in the case of genes A and Pn, which are located on linkage group III in japonica, showed a decrease. Probably, the relative positions of these two genes are quite different in indicas, although they are located in the same linkage group in japonicas. It appears from the data that the influence of mutagens on recombination values for genes located on different linkage groups is mediated through their effects on the segregation pattern. Treatments given at the pre- meiotic stage (zygotene-pachytene) led to either an increase or decrease in the recombination percentage. In the tillers, which were mostly'in the earlier stages, there was a decrease in the values thus indicating the influence of the mutagens to be stage specific. SWAMINATH AN et al. 31

TABLE I. SALIENT FEATURES OF THREE HIGH-YIELDING RICE VARIETIES

Variety Salient features

O. sativa subsp. indica *

(a) IR-8 A semi-dwarf (90-105 cm) high yielding (7-10 tons)

variety with response to high levels of N2; moderately,early maturing (110-120 days); photo- insensitive, moderate resistance to blight and tungro but susceptible to blast; low milling recovery (46.5), Developed from the cross Dee-geo-woo-genV^Peta.

(b) IR-5 A semi-dwarf but taller than IR-8 (130-140 cm); " late in maturity (130-145 days); weakly photosensitive; resistant to blight and susceptible to some races of blast; moderately lodging and better cooking quality. Developed from the cross.

O. sativa subsp. japonica

Tainan-3 Ideal plant type, but taller than the indica dwarf (120 cm); high yielding; photo-insensitive; high degree of resistance to bacterial blight; early maturing (110-115 days); poor cooking quality and hard threshing.

TABLE II. PERCENTAGE OF SURVIVAL IN IR-

No. of seeds No. of plants Percentage of Mutagen Dose treated which survived lethality

Control 200 50 0.00

Gamma rays 15 kR 500 82 34.00 30 kR 500 59 51.80

EMS 0;33% 500 102 18.40 0.50% 500 90 28.00

NMU 0.01070 500 47.20 0.015% 500 61 51.20

NG 0.005% 500 64 48.80 0.010% 500 59 52.80 32 SWAMINATHAN- et al.

TABLE III. FREQUENCY OF CHLOROPHYLL MUTATION IN IR-8

No. of Mj No. ofM2 families Percentage of Mj Mutagen Dose families segregating for plant progenies • studied mutagen segregating

Gamma rays 15 kR 82 29 35.36 30 kR 56 21 37.50

EMS 0.33% 90 24 26.66 0.50% 81 20 24.69

0.010% 66 13 19.69 0.015% 61 12 19.67

0.005% 59 22 37.28 0.010% 64 34 53.12

TABLE IV. EFFICIENCY AND EFFECTIVENESS OF IR-8

^ ^ D Efficiency Effectiveness Mutagen ose Mutated plants/lethality Mutated plants/kR or Cone, xtime

Gamma rays 15 kR 1.028 ' 2.357 30 kR 0.724 1.250

EMS . 0.33% 1.448 10.098 0.50% 0.882 6.172

NMU 0.010% 0.417 246.125 0.015% 0.384 163.916

NG 0.005% 0.764 ' 932.000 0.010% 1.006 664.000 SWAMINATH AN et al. 33

TABLE V. PERCENTAGE OF MUTATIONS IN ' SEGREGATING FAMILIES OF,IR-8

/ Mutagen Dose Segregation ratio

Gamma rays 15 kR '8.64; : 30 kR 15.36 .

EMS 0.33% ^ . .7.31 ' 0.50% ' 8.22

NMU 0.010% 6.76 0.015% 6.79

.. NG • ' 0.005% '•' 20.04 1 ' 0.010% 'i 22.45

TABLE VI. PERCENTAGE OF SURVIVAL IN NEUTRON TREATMENT

Treatment No. of seeds No. of seedlings Percentage of Variety (rads) germinated survived survival

IR-8. Control-. 500- " 360 ' 100

2400 1800 765 . 59

3000 1775, 1030 ; '79

Tainan-3 Control 500; 173 100

1800 , ., 1030 355"' '74

2400 1030' • , 366 • 77 - i

TABLE VII. FREQUENCY OF CHLOROPHYLL MUTATIONS (M2)

IR-8 .' ' Tainan-3 Fast-neutron

dose Percentage of r • Percentage of No. of spike No. of spike •. ° (rads) spike progenies ;. spike progenies progenies progenies - . r ° - segregating segregating

1800 . - 219 " '• 45.2

2400 385 28.6 148 31.1

. 3000 409 19.1- TABLE VIII. RELATIVE FREQUENCIES OF VARIOUS CHLOROPHYLL MUTATIONS (M2 )

Fast-neutron Frequency (in °jo) Variety dose .(rads) Albina Xantha Chlorina Alboviridis Striata Maculata Virido alba . Tigrina

IR-8 2400 .73.90 0.83 16.72 - ' ' 7.35 . 0.33. 0.33 • , 0.50 •

>. 3000 82.12 2.53 9.90 - - ' 4.95 - ,*• " ; 0.48

Tainan-3 1800 ' 44.70 - 8.6o . ; 35.04 5.60" ' '2.10 13.20 . - • '0.53 -

2400 67.50 2.50 24.02 3.80 1.90

TABLE IX. MUTAGENIC EFFECTIVENESS AND EFFICIENCY OF NEUTRON TREATMENTS

Fast-neutron - Effectiveness Efficiency Variety dose (Mutated spike/krad) (rads) Mutated spike/lethality • Mutated spike/pollen sterility Mutated spike/seed sterility

1R-8 2400 11.9 0.698 0.445 0.824

3000 8.0 0.910 0.284 0.461

Tainan-3 1800 18.8 1.738 0.644 1.232

2400 12.9 1.352 0.388 0.730 SWAMINATH AN et al. 35

TABLE X. PERCENTAGE OF ALBINAS IN SEGREGATING FAMILIES (ALBINA) IN- M., ' '-' . . ..; : •

-.Fast-neutron No. of albina No. of normal Segregation , Variety - dose seedlings green seedlings ratio ' (rads) i .

'•IR-8 2400 72 209 - 25.6

' 3000 100 288 • 25.8 '

Tainan-3. " * 1800 ,117. 1302 8.2

' . 2400 64 • 341 ,'15.8

TABLE XI. FREQUENCY OF MUTATIONS IN TAINAN-3 .

Treatment Chlorophyll ' Viable

Control 0 0

Gamma rays (kR)

.20 42.79 78.69 30 • 48.00 ; - 80.00 40 No survival

EMS (%) .

0.2 47.83 72.73

0.3 47.50 74.00

0.4 48.84 41.68

NMU {%)

0.025 35.16 44.45

0.050 39.18 45.00

0.100 50.05 57.09 36 SWAMINATHAN- et al.

TABLE XII. FREQUENCY OF MUTATIONS-IN IR-8

Mutation frequency (°/o M2 families) Treatment Chlorophyll Viable

Control 0 - 0

Gamma rays (kR)

20 43. 73 59 37

30 45. 94 60 93

40 42. 50 57 50

EMS (%)

- . . 0.2 23 08 53 84

0.-3 ' - " ' 27 45 62 74

0;4 38 00 - 58 00

' 0 .'5 33 33 60 00

NMU •(%)

0.025 34 92 44 00 .

0.050 58 02 56 68

0.100 49 10 60 64 SWAMINATH AN et al. 37

TABLE XIII. FREQUENCY OF indica MUTANTS IN TAINAN-3'

Per 100' Mx Per 100 Mt • Treatments plant progenies' spike progenies

Control ,

Gamma rays'(kR) ''

20 (with husk) 14.75 3.64

20 (without husk) 18.75 5.22

30 (with husk) 14.00 3.04

30 (without husk) 12.50 . 2.50

EMS (%)' '

0.200 (with husk) , '9.09 1.96

0.200 (without husk) 11.11 2.22

0:300 ' 10.00 2.55

0.400 11.36 5.44

NMU (%)• '. ,

0.025 ^ '

• 0.050 (with husk) '

. . 0.050 .(without husk) 6.67 2.44

0V100 ' 38 .SWAMINATHAN et.al.

TABLE XIV.' TYPES OF VIABLE MUTATIONS

Variety Hab'it Leaf Panicle Grain

IR-8 • • 1. Dwarf 9. Broad leaf 17-. Open -habit 22. Fine

'2. Tall 10. Narrow leaf 18. Compact habit 23. Coarse

. 3. Bushy • 11. Boat leaf 19. Muffled ear 24. Japonica

4. Grass clump 12. Brittle 20. Hard threshing 25. Glabrous .

5. Spreading 13. • Rolled flag 21. Bifurcated ear 26. Beaked leaf

6. Profuse tillering 14. Dark green 27. Elongated palea

7. Early ' "• . •15. Chlorina 28. Poly-husked

8. Late 16. Black spotted 29. Double kernel leaf 30. Large sterile glume

Tainan-3 1. Dwarf 12. Broad leaf - 19. Open habit 26.. White

2. Semidwarf -13: Narrow leaf 20. Compact habit '27. Indica

3. Grass clump 14. Boat leaf '21. Clustered habit 28. Bold

. 4. Bushy 15. Rolled leaf 22. Muffled ear 29. Short '

5. Spreading 16; 'Dark green 23. - Free threshing 30. Awned

6. Trailing" type • 17. Chlorina 24. Shattering 31. Glabrous

7. Profuse tillering Mi- Black spotted 25. Bifurcated ear 32. Beaked -

8. Thin stem ' 33. Poly-husked

9. Erectoides 34. Elongated palea

10. Early 35.- Larger sterile glume

11. Late 36. Sessile ' SWAMINATHAN etal. . .• 39

'TABLE XV. EFFECTS OF PRE-SOAKING' -

Germination Mutagen Pre-soaking • , Survival Variety and dose (in hrs) No. ofseeds percentage . - Percentage . sown

t Tainan-3 .. . Control 16 ' . 500 •• 90. 0 41.6

5<7o dMSO 16 '500 ' . 36. ,0 • 29.8

EMS + 5% dMSO 16 500 , .35. ,0 28.0

17 • . 500 , ; ' 32.. 0 20.8

18 . 500 • ' '31.,4 " ' 14.4

19 '' 500 - '. - •24. .4 22.2

20 • 500 ;; ' 29.. 2 19.6 ' ^ ' • 'i Tainung-65 Control . 16 . , *' 500. :',)••; •• '78,.4 ' . '"••.35.6

EMS 16 500 v' ' 44,, 0 .'• - <- ' -

•' 5% dMSO 16 '-50b '• 41. ,6. '. 29.0

1 % EMS + S°Jo dMSO 16 r5oo ' '37. .6 ' ' 30.2

17 500; • ' 36.. 0 ' - 25.8'

18 500 39.• o-' ' .•• 33.4

19 - 500 • 35, 0 14. 0

20 ' •. . 500 34,, 0 20.8 40 SWAMINATHAN- et al.

TABLE XVI. PERCENTAGE OF SURVIVAL AFTER EMS, dES AND dMSO TREATMENTS AFTER DIFFERENT PERIODS OF PRE-SOAKING (TAINAN-3)

Period of Percentage of survival pre-soaking (hours) EMS : - '' •1 d ES dMSO.

Control • " 87.00 87.00 - 87.00

8 V 69.3 96.0 . - 88.8

11 • 60.5- -59.2' 51.2

i4 60.5 79.8 45.8-

17 . - 47.8 55.2 51.4

18 "43.4 61.4 47.2

19 • 70.8 49.6 42.6

20 34.0 45.4 43.8

21 47.0 11.4 41.6

22 46.9 48.6 43.2

TABLE XVII. CHIASMA FREQUENCY IN CONTROL AND. X-IRRADIATED PANICLES OF japonica X indica HYBRID

Mean No. of No. of cells Material . Treatment chiasmata/ cell analysed • ± S. E.

' ' 1-45 XA-58

1. Control 50 24.06 ±0.15

2. Control 50 ' 24.18 ±0.14

3. 240 R (24 h) 100 '24.86 ±0.18

4. " " 480 R (24 h) 100 26.70 ±0.19

5. 1200 R (24 h) 100 25.13 ±0.13

6. 480 R (48 h) 100 25.09 ±0.12

7. " » 1200 R (48 h) 50 24.62 ±0.19

C. D. at 5% level for different combinations

1X3 = 0.738, 1x4 = 1.14; 1x5 = 1.07, 1 x6 = 0.604, 1 x7 = 0.867,1 x2 = 0.617 SWAMINATH AN et al. 41

TABLE XVIII. PERCENTAGE OF RECOMBINATION FOR DIFFERENT GENE COMBINATIONS WITHIN LINKAGE GROUPS; IN CONTROL AND AFTER X-IRRADIATION AND • MITOMYCIN C (MC) TREATMENT AT DIFFERENT FLOWERING STAGES

Recombination % , , Gene „, : Material . Treatment : combinations Pre-meiotic Post-stage Other branches

1-45 x A-58 CBwxxcbWx Control 35.08 35.08 35.08

240 R 40.00 40.00 37.47

„ 480 R • - 40.12 - 35.64

1200 R - 45.24 - 33.33

81.54 ' °A Pn xa pn Control 81.54 - „ 240 R 55.46 - 78.68

480 R . 48.57 - 81.42 „ 1200 R 65.64 _ - 69.64

0.001% MC 62.91 - 46.20 42 SWAMINATHAN- et al.

TABLE XIX. PERCENTAGE OF RECOMBINATION FOR DIFFERENT GENE COMBINATIONS BETWEEN LINKAGE GROUPS IN CONTROL AND AFTER X-IRRADIATION AND MITOMYCIN (MC) TREATMENT AT DIFFERENT FLOWERING STAGES

Recombination % Gene Material Treatment combinations Pre-meiotic Post-stages Other branches

N-45 X 1-6 PI X D6 pi Control 30.29 30. 30.29

240 R 36.75 31. 30.77.

480 R 44.79 23. 14.50

1200 R 34.14 19.99

0.001% MC 37.70 16.67

1-45 X A-58 Pr wx X pr Wx Control 34.88 34.88 34.88

240 R 16.00 37.69

480 R 39.33 52.65

1200 R ' 65.46 39.21

0.001% MC 59.21 39.99

Pn wx x pn Wx Control 38.50 38.50 38.50

240 R 30.29 19.99

480-R 35.43 51.04

1200 R 44.44 11,85

0.001% MC 37.59 38.23

A wx x a Wx Control 12.31' 12.31 12.31

240 R 16.00 14.04

480 R 16.09 12.70

1200 R 20.47 11.11

Pr Pn X pr pn Control 27.14 27.14 27.14

240 R 50.00 30.91

480 R 39.33 63.57

1200 R 48.60 48.57

0.001% MC 40.12 60.34

CB Pn x c pn Control 81.21 81.21 81.21

240 R 55.46 78.68

480 R 48.57 81.42

1200 R 65.64 ' 69.64

0.001% MC 62.91 46.20 SWAMINATHAN et al. 43

REFERENCES

[1] KONZAK, C.F. et al., in The Use of Induced Mutations in Plant Breeding (Rep. FAO/lAEA Tech. Meeting, Rome 1964) Pergamon Press (1965). [2] SIDDIQ, E. A., SWAMINATHAN, M.S., RiceBreeding with'Induced Mutations, Tech. Reports Series No.86, IAEA, Vienna (1968) 25.

DISCUSSION

G.T. SCARASCIA: What is the degree of synchronization of the meristematic nuclei of the presoaked seeds at the moment of the mutagen treatment? M.S. SWAMINATHAN: It is fairly high, about 75 - 80% under constant conditions of temperature, light and humidity.

HAPLOID RICE PLANTS IN MUTATION STUDIES

S. TANAKA Institute of Radiation Breeding, Ministry of Agriculture and Forestry, Ohmiya, Ibaraki-ken, Japan . . , ' .

Abstract

HAPLOID RICE PLANTS IN MUTATION STUDIES. Studies were made on chlorophyll-deficient' sectors and diploid-like sectors in haploid rice plants exposed to chronic gamma irradiation, and on germinal mutations in diploid strains derived from the haploid plants. The induction and elimination of somatic mutations.in haploid plants and the occurrence of drastic germinal mutations in diploid strains from haploid plants are discussed.

INTRODUCTION Haploid rice plants are easily distinguished from diploid plants by their miniature features and almost complete sterility. Many papers have b'een published on the morphological and cytological studies of haploid rice plants and the spontaneous diploid strains from haploid plants [1-4]. Furthermore, when haploid plants are treated with mutagens, induced mutations (most of which are recessive'to their wild types) are detected as mutated sectors on the irradiated haploid plants. By duplicating the .chromosome complement of haploid plants, pure diploid lines are obtained. Thus, haploid plants are suitable material for mutation studies. In the present experiments the appearance of somatic mutations, diploid-like sectors in haploid pla:nts and germinal mutations in diploid strains from haploid plants following chronic gamma irradiations were studied.

MATERIALS AND METHODS

One haploid. rice plant of Norin No. 8 has been propagated in the Institute of Radiation'Breeding since 1962, and 563 haploid plants originating from this, haploid plant were obtained in the spring of 1964. These haploid plants were exposed to chronic gamma irradiation in the gamma-ray field of the Institute of Radiation Breeding in 1964. The dose-rates and duration of exposure to gamma rays are shown in Table I. Growth inhibition was so severe after irradiation at dose-rates of 400 and'450 R/day that irradiation was discontinued after 60 days. • In 1964, chlorophyll-deficient sectors and diploid-like sectors were observed in haploid plants that.had been irradiated. Chlorophyll-deficient sectors were scored on the 25th of July and the 3rd of November 1964, 40 and 140 days respectively after the' start of the irradiation period. Chlorophyll-deficient sectors appeared in different sizes and those sectors which were larger than two thirds of a leaf length were recorded as mutated sectors. Plants or tillers with one or more leaves showing such chlorophyll-deficient sector(s) were recorded as mutated plants or tillers.

45 05

TABLE I. SCHEME OF CHRONIC GAMMA IRRADIATION OF HAPLOID PLANTS

Treatment ' 1 2 3 4 5 6 7 ' - 8 9 '. • 10 11 12

Duration of growth in the gamma-ray field (days) 142 69 69 142 ' 142 142 142 142 142 . 142 142 -

Irradiation times (days) 104 • 60 60 . 104 '104 . 104 104 .104 ' 104 104 104 -

Dose-rates (R/day) a - ' 500 450 400 350 • 300 250 200 150 100 , 50 25 0

Dose (.kR) 52.0 27.0 •24.0 36.4 ' 31.2 26.0 - 20.8 - 15.6 10.4 5.2 . 2.6 0 •

Note: Transplanting date: 5th June, 1964, ' Irradiation started on 15th June, 1964, and terminated on 15th October, 1964. ' • • Gtowth inhibition was so severe after dose-rates of 450 and 400 R/day, that irradiation -was discontinued 60 days after it was started. Although irradiationatadose-rateof500 R/day caused very severe growth inhibition irradiation was continued to investigate the lethality until-the autumn of 1964. - a R/day was a 20-h day due'to administrative.reasons. . * • - TANAKA 47

The occurrence of diploid-like sectors, which are easily, distinguished from haploid tissue by the size of the leaf, panicle, gimme, etc. were investigated on irradiated plants. Diploid-like panicles often produced seed(s). The number of panicles, both ha:ploid and diploid-like, were recorded and all the panicles with seed(s) were harvested in.the autumn of 1964. All the stems and leaves of the irradiated and non-irradiated haploid plants were cut off in September of 1964 and the stubbles were kept in a greenhouse until the spring of 1965. In 1965, observations were made on the chlorophyll-deficient sectors and diploid-like sectors in the haploid plants propagated vegetatively in the spring of 1965. When new sprouts emerged, shoots of the'haploid plants in the greenhouse were separated and transplanted from pots to an experimental field. Plants derived from each- of the irradiated plants from the preceding year were grouped as individual clonal strains. Chlorophyll-deficient sectors and diploid-like sectors observed on the above plants were larger than those on the progenitors in 1964. Observations on chlorophyll-deficient sectors were made on the 15th of August 1965, and on dipl'oid-like sectors on the 30th of October 1965 by the same methods as those used in 1964. All the diploid-like panicles with seed(s) were harvested and stored until the spring of 1967 together with the diploid-like panicles harvested in 1964. The pedigree method was used on germinal mutations of diploid strains derived from haploid plants. The original haploid plant had been maintained and vegetatively propagated in the -National Institute of Agricultural Sciences.' During the multiplication, ten spontaneous diploid strains were obtained. One of them has been used in mutation studies in the Institute of Radiation Breeding. " In the spring of 1967, 325 panicle progenies from irradiated haploid plants; one strain from non-irradiated haploid plants; ten spontaneous diploid strains maintained in the National Institute of Agricultural Sciences and one spontaneous diploid strain which has been used in the Institute of Radiation Breeding were sown in a field nursery. Owing to the high sterility of the panicles obtained from irradiated haploid plants and the severe inhibition of germination of the seeds obtained from these panicles, only 93 diploid strains from irradiated haploid plants consisted of more than five plants, and 52 strains consisted of less than four plants. The other 180 strains did not germinate. The 93 strains were transplanted from the nursery to an experimental field together with ten spontaneous diploid strains maintained in the National Institute of Agricultural Sciences, and one spontaneous diploid strain which has been used in the Institute of Radiation Breeding as a control. The control strains were planted in every tenth row for comparison. Any visible, changes- observed on these strains were recorded.

RESULTS

The radiation damage induced by. chronic gamma irradiation on haploid plants was similar to.that observed on diploid plants, .although the. radio-sensitivity of the'haploid plants was higher than that of the diploid plants. Growth inhibition was very severe in those plants that were irradiated at dose-rates of 500, 450 and 400 R/day; severe at TABLE II. FREQUENCIES OF PLANTS. AND TILLERS WITH CHLOROPHYLL-DEFICIENT SECTORS INDUCED BY CHRONIC GAMMA IRRADIATION OF HAPLOID PLANTS (July'1964)

t Dose-rates (R/day) ? 500 450 400 350 . 300 250 - 200 150 100 50 ' 25 0 Total

No. of plants irradiated _ 40 40 40 ' 40 40 " 40 40 40 . 40 40 •40 40 480 '

No. of plants with chlotophyll- 24 32 33 27 31 24 27 40 25 5 0 0 268 deficient sectors b

Frequencies of plants with chlorophyll- 60.0 80.0 82.5 67.5 77.5 60.0 67.5 100 62.5 12.5 0 0 deficient sectors (°jo)

No. of tillers irradiated 357 ; 267 222 439 414 402 550 599 475 •451 512 427 5133

No. of tillers with chlorophyll- .22 24 29 35 29 32 . 40 ' 91 39 9 0 0 • 350 deficient sectors

Frequencies of tillers with chlorophyll- 5.9 9.0 13.1 8.0 7.0 8.0. 7.3 15.2 8.2 • 2.0 0 0 • deficient sectors (^o)

a See Table I. , Chlorophyll sectors which were longer than two-thirds of the leaf length were recorded. TABLE III. FREQUENCIES OF PLANTS AND TILLERS WITH CHLOROPHYLL-DEFICIENT SECTORS INDUCED BY CHRONIC GAMMA IRRADIATION OF HAPLOID PLANTS (Nov. 1964)

Dose-rates (R/day) a 500 450 400 350 300 250 200 150 100 50 25 0 Total

No. of plants irradiated 28 32 40 38 38 40 40 40 40 38 40 39 ' 454

No. of plants with chlorophyll- 13 25 38 16 19 18 21 37 28 28 24 deficient sectors ^

Frequencies of plants with chlorophyll- ^ ^ g5_Q ^ 5Q_0 ^ ^ g2_5 7Q_0 73_? 60_Q ^ * g deficient sectors (70) ^

No. of tillers irradiated 503 340 1023 824 1136 996 1633 1489 1053 957 1013 916 11885 >

No. of tillers with chlorophyll- 21 45 126 34 31 30 40 113 50 68 50 4 612 deficient sectors

Frequencies of tillers with chlorophyll- 4.2 13.2 12.3 4.1 2.7. 3.0 2.4 7.6 4.7 7.1 4.9 0.4 deficient sectors (%)

See Table I. See Table II.

cd TABLE IV. FREQUENCIES OF HAPLOID PLANTS WITH CHLOROPHYLL-DEFICIENT SECTORS, VEGETATIVELY PROPAGATED AFTER CHRONIC GAMMA IRRADIATION (August, 1965)

Dose-rates (R/day) a 500 450 400 . 350 300 250 200 150 100 50 25 0 Total

No. of tested plants 484 - - 801 1098 942 1592 1468 1002 963 992 892 10234

No. of plants with chlorophyll- 24 - - 20 20 3 21 9 5 3 7 0 112 deficient sectors

Frequencies of plants with chlorophyll- _ _ 2_5 ^ Q_3 ^ 0_g Q_5 Q_3 Q_7 0_Q deficient sectors (^o)

Plants irradiated at dose-rates of 450 and 400 R/day were not propagated because their growth was too severely inhibited. a See Table 1. TANAKA 51 dose-rates of 350 and 300 R/day; marked at a dose-rate of 250 R/day; slight at a dose-rate of 200 R/day; very slight at dose-rates of 150 and 100 R/day; and at dose-rates of 50 and 25 R/day the growth of irradiated plants was almost normal. Chlorophyll-deficient sectors appeared two or three weeks after the start of the irradiation period. The higher the dose-rate the earlier the appearance of such sectors. The size of the chlorophyll-deficient sectors increased with growth and increase of total dose. The frequency of the plants and tillers with chlorophyll-deficient sectors observed in July and November of 1964 are presented in Tables II and III. As shown in these tables, the frequency *of the plants or tillers with chlorophyll-deficient sectors observed in July, 60 days after the start of irradiation at dose- rates of 50 and 25 R/day, were distinctly less than those irradiated at dose-rates of 100 R/day arid above. However, observations made in November of 1964, 160 days after the start of the irradiation period, indicated that there was no difference between the frequency of the plants or tillers with chlorophyll-deficient sectors after irradiation at high dose-rates and low dose-rates. The relationship between the frequency of chlorophyll-deficient sectors on plants or tillers and the dose-rate or total dose is more complicated when growth takes place under chronic gamma irradiation than under acute irradiation. The induction and elimination of mutations in cells of growing points, the death of cells in growing points, the differential radio-sensitivity of those cells and other factors may be related to the appearance of chlorophyll-deficient sectors on chronically irradiated haploid plants. Table IV shows the frequencies of the haploid plants that formed chlorophyll-deficient sectors after they were vegetatively propagated in 1965, six months after the end of the irradiation period. Observations made in August 1965, nine months after the end of irradiation.period, revealed a tendency for the frequency of the plants with chlorophyll- deficient sectors to increase with increasing dose-rates or total dose. This relationship was not observed in the plants treated with chronic gamma irradiation in 1964. The size of the sectors on vegetatively propagated haploid plants was generally larger than that of sectors observed on haploid plants under chronic irradiation. The frequency of haploid plants with diploid-like sectors, after chronic gamma irradiation in 1964 and vegetative propagation in the spring of 1965, are summarized in Tables V and VI. The frequency of haploid plants with diploid-like sectors increased with increasing dose- rates in 1964, and these sectors were not observed on the haploid plants exposed to gamma rays at dose-rates of less than 250 R/day in 1964. However, the frequency of haploid plants with diploid-like sectors after vegetative propagation in 1965 was not particularly high after irradiation at liigh dose-rates. The size of the diploid-like sectors (i.e. the proportion of diploid-like panicles in the total panicles of the plants) growing in the gamma-irradiated field increased remarkably with growth. This seems to suggest that diploid-like sectors are more radio-resistant and develop faster than haploid tissue under irradiation at high dose-rates. The size of the diploid-like sectors under irradiation in 1964 showed different fertilities ranging between 0% and 90%, the average being 30% in most cases. After vegetative propagation in 1965 most of the diploid- like panicles produced seeds and their fertility was more than 80%. CJl to

TABLE V. FREQUENCIES OF HAPLOID PLANTS WITH'DIPLOID-LIKE SECTORS INDUCED BY CHRONIC GAMMA IRRADIATION (Oct. 1964)

Dose-rates (R/day) a 500 450 ' 400 350 300- 250' 200 150 100 50 25 0 Total

No. of plants which survived 28 32 . 40 38 38 '40 40 40 40 38 40 39 453

No. of plants with diploid-like 1 3. 3 " 2 1 0 0 0 0 0 0 0 10 sectors

Frequencies of plants with 3.6 9.4 7.5 5.3 2.6 ------diploid-like sectors (°Jo) -

No. of tillers which survived 503 340 1023 824 1136 998 1633 1489 1053 957 1013 916 11885

No. of tillers with diploid-like 43 15 ' 38 7 6 0 0 0 0 .0 0 0 109 sectors

Frequencies of tillers with 8.5 4.4 3.7 0.8 0.5 ------diploid-like sectors (%)

Frequencies of diploid-like tillers

in the plants showing 42.9 26.3 60.3 13.3 37.5 ------diploid-like sectors (<7o) ^

No. of panicles with seeds 10 0 0 0 1 0 0 0 . 0 0 0 0 11

a See Table I. k Plants which sprout both haploid-like tillers and diploid-like tillers. TABLE VI. FREQUENCIES OF HAPLOID PLANTS WITH DIPLOID-LIKE PANICLES, VEGETATIVELY PROPAGATED AFTER CHRONIC GAMMA IRRADIATION (Oct. 1965)

Dose-rates (R/day) a 500 350 300 250 200 150 100 50 25 0 Total

No. of clonal strains propagated 28 38 38 40 40 40 40 38 40 39 453

No. of clonal strains which segregate 10 11 1 1 1 1 1 1 9 plants with diploid-like panicles

No. of plants propagated ^ gQ1 iQgg g42 ^ ^ ^ gf;3 ggz ggg i(m3 vegetatively

No. of plants with 10 1 6 4 5 " 1 3 4 4 2 40 diploid-like panicles

No. of diploid-like panicles 73 1 145 42 36 1 55 37 28 11 429

No. of panicles with seeds 64 1 130 31 22 1 47 5 24 1 265

a See Table I. Plants irradiated at dose-rates of 450 and 400 R/day were not propagated because their growth was too severely inhibited. TABLE VII. MUTATIONS IN M2 DIPLOID STRAINS DERIVED FROM DIPLOID-LIKE PANICLES

OF IRRADIATED HAPLOID PLANTS (Mx)

Dose-rates (R/day) a 500 350 300 250 200 150 100 50 25 0 Total Control b Spontaneous*3

No. of panicles with seed(s) in 1964 10 0 1 0 0 0 0000 11

No. of panicles with seed(s) in 1965 64 1 130 31 22 1 47 5 24 1 326

No. of panicle progenies sown 74 1 131 31 47 337 in 1967

No. of surviving panicle progenies 53 0 82 13 153 10 >H No. of strains tested c 29 0 54 7 93 10 >Z

No. of strains which segregate > 22 0 4 28 mutant plants

No. of strains fixed 0 50

No. of strains not tested ^ 28 60

See Table I. k Control and spontaneous diploid strains were obtained from spontaneous duplication of chromosome complement of the original haploid plant during vegetative propagation of haploid plants in the National Institute of Agricultural Sciences, and the control strain had been used in mutation studies at the Institute of Radiation Breeding. c Progenies of diploid-like panicles consisted of five or more plants. ^ Progenies of diploid-like panicles consisted of four or less plants. Plants were grown in pots without observation and one panicle from each plant was harvested to raise M , strains. TANAKA 55

The visible drastic changes observed in diploid M2 strains derived from irradiated haploid plants and spontaneous diploid strains originating from the same haploid plant are recorded in Table VII. Fifty-eight strains without any drastic changes and seven strains that were pale green seemed to be true-bred strains in the M2 generation. Twenty-six strains segregated plants with one of the drastic mutant types, such as dwarfing, short-culm and erect panicle, and the other two strains segregated both dwarf plants and plants with erect panicles. All the chlorophyll mutants observed in 1967 were pale green. Most of the morphological mutants were dwarfing and a few of them were short culms and erect panicles. In one strain, early heading plants (ten days earlier than the control) were segregated. Plants with changes of fertility were relatively, rare. The segregation frequency of these mutant plants within the mutant strains was nearly 25% in most cases, which suggests a chimeric structure of the panicles from which these- strains were derived, or inductions of mutations after duplication of chromosome complement of haploid plants.

CONCLUSIONS

The frequency of the chlorophyll-deficient sectors did not show a clear dependence upon dose-rates or total dose. This suggests that many factors are involved in the, appearance of mutated sectors on chronically irradiated haploid plants. These factors,should be investigated to establish effective methods of utilization of haploid plants in mutation studies. The size of the diploid-like sectors under chronic gamma irradiation at high dose-rates increased remarkably with growth. This phenomenon may be caused by differential radio-sensitivity between diploid sectors and haploid tissue. Diploid strains derived from irradiated haploid plants unexpectedly segregated drastic mutants. Fixed mutant strains with drastic changes such as dwarfing, short culm and erect panicle were not observed in the

M2 generation. This fact seems to suggest that the most drastic mutations induced in haploid plants are not transmitted to the next generation and that these drastic mutations observed in the M2 generation were induced after duplication of the chromosome complement of the haploid plants. These phenomena are very important both theoretically and economically.

ACKNOWLEDGEMENTS

The author wishes to thank Dr. H. Akemine for his useful suggestions and Dr. T. Kawai for his helpful advice.

REFERENCES

[1] MORINAGA, T., KURIYAMA, H., AOKI, M., A sterile and extremely dwarf mutant occurred in a theoretically pure line of rice, Jap. J. Genet. 18 (1942) 297. [2] HU, C.H., Karyological studies in haploid rice I, lap. J. Genet. 32 (1957) 28. [3] HU, C.H., Karyological studies in haploid rice II, Jap. J. Genet. 33 (1958) 296. [4] HU, C.H., Karyological studies in haploid rice III, Jap. J. Breed. 9 (1959) 135. 56 TANAKA

DISCUSSION

N. YAMADA: What practical applications could result from your research, or is it still too premature to draw conclusions?

S. TANAKA: In the F2 generation one can obtain pure and fixed diploid lines by duplicating the chromosome complement of the haploid plants. Such pure homozygous plants are very useful in breeding. It is too early to draw conclusions concerning the application of induced mutations in this work. B. SIGURBjdRNSSON: I have heard of your successful results in inducing an increase in the protein content of rice. Could you comment on that? S. TANAKA: Yes, protein analysis has been carried out on a number of our mutants. Some of them produce twice the protein content of the mother variety. INDUCED.MUTATIONS OF RICE FOR SHORT-CULM SELECTIONS

IN M2 GENERATION""

J.H. REE Yungnam Crop Experiment Station, Office of Rural Development, Milyang, Korea

Abstract

INDUCED MUTATIONS OF RICE FOR SHORT-CULM SELECTIONS IN M2 GENERATION. Seeds of a leading rice variety Palkweng, japonica , were treated with X-rays and thermal neutrons to obtain mutations having short culm, earliness, resistance to lodging and blast disease, and a high yielding ability. 507 plants were selected for short-culm length; on the average they were also shorter in panicle length, lighter in weight of panicles, less in 100-grairi weight and earlier in days to heading. There was no strict correlation between culm length and panicle length. Some plants had longer panicles in spite of a distinct reduction in culm length, but the number of panicles, weight of panicle and days to heading were positively correlated with culm length in the selected plants. The length of each internode from the base of the panicle down to the basal internode was gradually reduced. The lodging index was lower than that of the original variety, and culm length was positively correlated with lodging index.

INTRODUCTION

The study reported here was made'on a leading rice variety Palkweng, japonica. Palkweng is an old variety with a long culm, medium tillering and medium maturity, that is susceptible to lodging, moderately susceptible to blast disease, but high yielding and with good eating quality. It is grown on approximately 34% of the total area of paddy fields in south-eastern Korea. The seeds of Palkweng were treated with X-rays and thermal neutrons to obtain mutations having short culm, earliness, resistance to lodging and blast disease, and still retain the high-yielding ability. The induction of short-culm mutations by radiation'treatment may be one of the promising methods for rice breeding as reported also by Beachell, Kawai, Tanaka and Toriyama [1-6] . Since short-culm plants obtained in this experiment have not yet been thoroughly examined, especially for their desirable agronomic characters, this experiment may also be worth continuing. ' • .

EXPERIMENTAL METHOD

This experiment was initiated in 1966 and continued through 1967. In 1966, seeds of Palkweng were given five different treatments with 20 000 and 25 000 R of X-rays, and 5X 1012/cm2, 10X1012/cm2, 15 X 1012/cm2 of thermal neutrons at the Institute of Atomic Energy Agency, Seoul, Korea.

* This work was partly supported by the IAEA under Research Contract No. 547/RB.

57 58 REE

Mi plants were harvested separately. Data were recorded on germination and fertility of the Mi panicles. In 1967, seeds of the second generation (M2) were sown directly on a raised seed bed on May 10, and all seedlings were transplanted in a paddy field on June 25. All the plants were grown spaced in 24X 15 cm lattices and fertilizers were applied at 120-80-80 kg/ha of N, P205 and-K20, respectively.

• In the M2 generation, progenies of three or four panicles of each M! plant were grown as panicle progenies in rows. 3900 panicle progenies, with a total of 228 000 plants, were planted. The original variety Palkweng was planted as control at ten-row intervals. During the growing period variations were observed in the agronomic characters, such as heading date, plant height, tillering and plant type. All plants that headed earlier than the original variety were tagged. At maturity, the degree of sterility, grain size and.shape, spikelet density, and other characters of M2 generation.plants were also observed simultaneously with the character of short culm. Three different criteria were used in selecting short-culm variant types in the M2 generation; (i) distinctly shorter culm plants having other desirable agronomic characters; (ii) slightly shorter culm plants having other desirable agronomic characters; and (iii) plants which were regarded as belonging to the short-culm variant type but showing only slight differences in agronomic characters. Those plants selected in the M2 generation will be tested in the subsequent generation in 1968. The extremely short- culm plants with distinctly poor yielding characters were discarded in the field selection. Laboratory tests of bending moment, breaking strength and lodging index were made on plants randomly sampled from the field selections for short culm, as a basis for detecting lodging resistance. For analysis

TABLE I. RICE PLANTS AND LINES TESTED AND SELECTED FOR SHORT CULM IN THE M2 GENERATION IN 1967

Number of % of the Radiation plants dose Lines Plants Lines Plants selected tested tested selected selected

X-rays 20 kR 1200 72 000 61 265 0.37

25 kR 900 60 000 51 133 0.22

Thermal neutrons 5xl012 700 36 000 29 0.08 n(th)/cm!

10 x 10*' 700 36 000 25 36 0.10

15 X 101: 400 24000 15 44 0.18

Total 3 900 228 000 191 507 0.22 REE 59 of yield components, such as number of panicles per plant, weight of panicle per plant and weight of 100 grains, were measured. Data obtained from the M2 generation plants were always compared with those from the original variety.

RESULTS.

In the Mi generation, 1405 plants out of 17 901 were screened in 1966, as presented in the Report of Yungnam Crops Experiment Station [7].

Out of 1300 M2 short-culm plants selected on the basis of observations in the field, 507 plants were re-selected in the laboratory by measuring certain agronomic characters. In this re-selection, those plants having sterility or malformation and other characters which clearly impair yielding ability were discarded. It was found that the treatment with 20 000 R of X-rays was the most effective in shortening the culm in this experiment (Table I).

The 507 plants selected in the M2 generation which had a short culm and normal fertility were studied for other desirable agronomic characters. As shown in Table II, the mean culm length of the 507 plants selected was 80.8 cm compared with 96.6 cm for Palkweng. The mean panicle length was 17.9 cm compared with 18.8 cm for the original variety. How- ever, the ratio of the panicle length to the culm length (PL/CLX 100) increased by 14% in the selected plants compared with the original one. This would suggest that panicle length does not always decrease pro- portionally to the shortening of the culm length. On the other hand, the mean value for the number of panicles per plant was 10.5 compared with 9.4 for the original one. However, the mean value for the panicle weight per plant was 22.5 g compared with 24.0 g for the original variety, and the weight of 100 grains was 2.5 g compared with 2.7 g for the original variety. These data indicate that the reduction in weight of the panicles

TABLE II. MEAN VALUES AND RATIOS OF CULM LENGTH, PANICLE LENGTH, NUMBER OF PANICLES, PANICLE WEIGHT PER PLANT, WEIGHT OF 100 GRAINS, AND DAYS TO HEADING OF

507 M2 PLANTS SELECTED FOR SHORT CULM COMPARED WITH THE ORIGINAL VARIETY IN 1967

Panicle Culm Panicle Ratio of Wt. of No. of weight per Days to length length panicle/ 100 grains panicles plant (g) heading (cm) (cm) culm length (g)

Original 96.6 18.8 19.5 9,4 24.0 2.7 104; 3 variety, Palkweng

Selected 80.8 17.9 22.2 10.5 22. 5 2.5 99.5 plants of M 2

Ratio of the 84 95 114 112 94 93 95 original variety TABLE III. PERCENTAGE OF PLANTS WHICH DEVIATE FROM THE CONTROL WITH REGARD TO CULM AND PANICLE LENGTH, NUMBER AND WEIGHT OF PANICLES PER PLANT, AND 100-GRAIN WEIGHT AFTER

SELECTION FOR SHORT-CULM LENGTH IN M2 (Total No.of plants, 507)

Culm Panicle No. of Wt. of 100-grain Classification3 length length panicles panicles weight Deviation from control 07°) (%) (%) (%) (%)

significantly 42 0 0 0 53

keduced

slightly 55 16 1 16 17

Little difference 3 52 83 76 22

slightly 0 27 13 ' 7 3

significantly 0 5 3 1 5

Mean and 96.6 ± 5.0 18.8 ±1.5 9.4± 2.2 24. 0±6.2 2.69 ±0.07

standard deviation (cm) (cm) (g) (g)

a Based on mean value with standard deviation; little difference falls within ± 1 S. D., slightly reduced or increased fall within ± 2 S.D., and significantly reduced or increased fall within ± 3 S.D. REE 61

is affected by the reduction in the percentage of fertility and the kernel weight. In the number of days from seeding to heading, the selected plants for short culm headed, on the average, about 5 days earlier and the earliest plants headed 12 days earlier than the original variety. Table III shows the frequency distribution in per cent of four characters of the 507 plants selected for short culm. In culm length 42% were significantly shorter, 55% were slightly shorter and 3% showed little difference compared with the original variety. In panicle length, 27% were slightly longer and 5% were significantly longer. On the other hand, 16% were slightly shorter and the rest showed little difference. However, in the number of panicles per plant, 3% of the 507 selected plants showed a significant increase, 13% a slight increase, 1% a slight reduction and the remainder showed little difference. In weight of panicles per plant, 1% were significantly heavier and 7% were slightly heavier, whereas 76% showed little difference and 16% were slightly lighter. It was shown that the weight of 100 grains in 17% of the 507 selected plants was slightly lighter and 53% was remarkably lighter compared with the original, whereas only 3% were slightly and 5% were remarkably heavier, respectively. As presented in Fig.l, the plants selected for short culm showed a smaller variation for panicle weight per plant than the original variety.

PANICLE WEIGHT (g)

FIG.l. Frequency curves for culm length and weight of panicles of plants selected for short culm in Mz, compared with the original variety. 62 REE

In the plants selected for short culm, the correlations between culm length and panicle length, number of panicles, and weight of panicles per plant are presented in Figs 2, 3 and 4. No correlation was found between culm length and panicle length. Some plants had longer panicles in spite of a distinct reduction in culm length. On the other hand, the number of panicles was positively correlated with the culm length, with a coefficient of 0.1582. The weight of the panicles per plant was also positively correlated with the culm length, with a coefficient of 0.4081, both significant at the 1% level. In general, the weight and number of panicles per plant seemed to decrease with decrease in culm length. Some exceptional plants, however, had heavier panicles, more panicles per plant and longer panicles in spite of a distinct reduction in culm length. These results are similar to those obtained by Kawai [2], Such a combination of characteristics is highly promising in practical rice breeding. Therefore, it is advisable to continue the tests on the yield characters mentioned previously so as to determine whether they will persist in subsequent generations, similar to reports by Tanaka [3]. From the 507 plants selected for short culm, 111 plants were sampled at random to determine lodging resistance. The length of internode, bending moment ( = culm length (cm)X tiller weight (g)), breaking strength and lodging index (= bending moment/breaking strengthX 100) were measured. The results in Table IV show that, in general, the length of each internode from the base of the panicle down to the basal internode was gradually reduced, and that particularly N3 (= the fourth internode from the base of the panicle) and N4 (= the fifth internode) internodes, which are closely related to resistance to lodging, were remarkably shorter than those in the original variety.

26- 1

12 2 1

2 115 12 24" 1 1 1 2 4 2 111 22- 4 3 2 6 4 10 3 6 5 1 3 3 5 6 .9 13 6 5 8 20- 2 6 10 9 9 9 12 12 11 1 3 9 10 12 8 12 15 1 2 11 4 10 11 7 4 9 2 1 7 2 5 10 3 1 16- 2 1 6 3 7 3 1 3 2 K- 2 1 2

63 75 79 83 87 91 67 71 CULM LENGTH (cm)

FIG.2. Correlation between culm length and panicle length of selected plants in M2. REE 63

24- 22-

18-

16-

7 2 8 10 4 12 12 13 10 12 8 11 16 16 9 8 7 IE 10 IE 10 8 9 7 12 1 7 6 7 1 2 1 1 1 1 1

63 67 79 83 67 CULM LENGTH (cm)

FIG. 3. Correlation between culm length and number of panicles per plant of selected plants in M2

61 -

57 •

53 " 49 -

45 "

3 41 -

2 37' a. x 33 " 1 2 2 2 3 1 4 1 1 6 3 2 1 2 3 1 3 4 6 2 6 1 1 1 4 6 8 11 E e 9 3 1 2 2 4 5 7 E 9 7 11 7 5 3 4 4 5 7 4 15 14 21 10 9 2 3 3 3 3 8 14 15 15 17 12 9 7 b 1 2 7 E 9 7 10 14 E 4 2

1 63 66 69 72 75 78 81 84 87 90 93 96 CULM LENGTH (cm)

FIG.4. Correlation between culm length and weight of panicles per plant of selected plants in M2. TABLE IV. LENGTH OF CULM AND INTERNODE, BENDING MOMENT, BREAKING STRENGTH, AND LODGING

INDEX OF 111 M2 PLANTS SELECTED FOR SHORT CULMS

Length of internode (cm) No. of Culm Bending Breaking Lodingj plants iength moment'3 strength0 (g) index tested (cm) N? N, N, N3 N4 Ns ' ' (g x cm)

Original 100 100 100 100 100 100. 100 . 100 100 100

variety, Palkweng 111 (98.2) (41.5) (21.4) (15.5) (11.9) (6.1) (2.0) * (1. 087) (981) (110)

Short-culm 111 77 94 85 79 46 25 25 . 67 129 52 Mj plants

Note: Figures in brackets show the actual mean values.

aNQ = The.first internode from the base of the panicle down to the basal internode.

^Bending moment = Plant height x tiller weight.

CBreaking strengths Tested with a 5-cm segment taken from the 4th internode.

d . , Bending moment Lodging index = rr"0 r x 100. ° ° Breaking strength REE 65

The bending moment of selected plants for short culm was less, whereas breaking strength was'heavier than that of the original variety,' and accordingly the lodging index was 57.5 compared with 110.0 for the original variety. The data in Fig.5 show that culm length and the lodging index were positively correlated with a coefficient of 0.5290,• significant at the 1% level. These results indicate that the plants selected for short culm had resistance: to lodging.

120

£ 100-

80 - • ••.* •. ••"• w*Jf*t o . • V o o 60

60 65 70 75 80 85 CULM LENGTH ( cm)

FIG. 5. Correlation between culm length and lodging index of 110 selected plants in M2 generation.

In this experiment, the lodging index appeared to be a fairly good indicator of the plant type, tall plants vs. short ones. Most of the short- culm plants have an advantage over the tall ones in their resistance to lodging. These results agree well with those given in Ref. [8].

CONCLUSIONS

In the short-culm plants selected in the M2 generation, other agronomic characters were changed simultaneously. The mean culm-length of the 507 plants selected was 81 cm compared with 97 cm for the original variety. The plants selected were shorter in panicle length, less in weight of panicles per plant and kernel weight, earlier in days to heading and shorter in culm length. The variation in culm length and other desirable characters showed a wider range than in the original variety. No correlation was found beween culm length and panicle length. • Some plants were found in which the panicle length'was not shorter in spite of a distinct reduction in culm length. The number and weight of the panicles per plant were positively correlated with the culm length. Some exceptional plants, however, had heavier panicles and more panicles per plant. . : The results obtained in this experiment indicate that each internode length of the plants selected for short culm was gradually reduced between the base of the panicle and the basal internode. In particular, the N3 and N4 internodes, which are closely related with resistance to lodging, were remarkably shorter than those in the original variety. The lodging index was lower than that of the original variety and the culm length was positively correlated with the lodging index. These results indicate that the plants selected for short culm had resistance to lodging. Such a combination of characters should give a very important reason for practical rice mutation breeding by means of radiation, as also reported by Kawai and Tanaka [2, 3]. As suggested in the recommendations 66 REE of Ref.[ 9], the fact that a mutant strain possesses positive and negative characters with regard to its usefulness in plant breeding is not always due to pleiotropic action of a single mutant gene. It can also be due to the compound effect of two or more neighbouring absolutely linked genes, which changed simultaneously during the mutagen treatment. The progenies selected for short culm having desirable agronomic characters must be tested in subsequent generations for the possibility of eliminating factors which impair yield components. In further studies in this project, not only the short-culm variants, bat also the slightly shorter ones which were not changed in other desirable agronomic characters, will be benefitial for practical breeding in rice.

REFERENCES

[1] BEACHELL, H.M., Newsletter (FAO) 6 (1957) 18. [2] KAWAI, T., SATO, H., MASIMA, I., "Short-culm mutations in rice induced by P3!", Effects of Ionizing Radiations on Seeds (Proc. Symp. Karlsruhe, 1960) IAEA, Vienna (1961) 565. [3] TANAKA, H., Studies on artificial induced mutation in rice breeding. The report of gamma field in Japan (1968) 83. [4] TORIYAMA, K., FUTSUHARA, Y., Studies on rice breeding by means of artificially induced mutation. II. The breeding of the high yielding strain Fu. 53 by irradiation treatment with P32, Jap. J. Breed. 12 (1962) 148. [5] TORIYAMA, K., FUTSUHARA, Y., Studies on rice breeding by means of artificially induced mutation. IV. The breeding of the high yielding line Fr. 54 by X-ray treatment, Jap. J. Breed. 12_ (1962) 263. [6] TORIYAMA, K., FUTSUHARA, Y., Breeding of new rice variety "REIMEI" by gamma-ray irradiation, Jap. J. Breed. 17 (1967) 85. [7] Yungnam Crops Experiment Station, The annual research report, Office of Rural Development (1966) 104. [8] IRRI, Annual Report of the International Rice Research Institute, Manila, The Philippines (1963) 22. [9] Panel of Mutations in Plant Breeding,' in "Scientific recommendations" (Proc. Panel Vienna, 1966) IAEA, Vienna (1966) 260.

' DISCUSSION

M.S.HAQ: Can you inform us about (a) the M2 population size, (b) the method of selection in Mj^ and M2, and (c) whether the variety used is late in maturing? J.H.REE: (a) The M2 population consisted of 17 900 plants, (b) In the Mi generation we discarded malformed plants; in the M2 population the main objective was to select for distinctly short culm and other desirable agronomic characters, and (c) the variety is medium to late. REE 67

RESULTS OF THE FAO/IAEA UNIFORM RICE MUTATION TRIALS IN 1967 FROM YUNGNAM CROPS EXPERIMENT STATION, MILYANG, KOREA

1. In 1967 a yield trial was carried out with 12 mutant strains and Palkweng as the check variety.

2. The seed was planted on 26 June and transplanted on 20 July, but it was too late for seeding in our climatic conditions.

3. The trial gave the following results: (a) Among the 13 varieties, four produced more tillers and seven attained earlier maturity than Palkweng, whereas three did not mature. (b) The culm length was shorter than that of Palkweng in nine varieties and longer in three varieties. All the varieties were non-lodging except Allorio-11. In particular, we observed that the mutant variety Allorio lamda was 25% shorter in culm length and non-lodging, and that Reimei was 13% shorter in culm length than the original varieties Allorio-11 and Fujiminori, respectively. (c) Only two mutants yielded more grain than the original variety. Allorio lamda yielded 15% more than Allorio-11 and Reimei yielded 4% more than Fu'jiminori. (d) In' general, disease was seldom observed and there was no incidence of insects or pests.

IMPROVEMENT OF RICE VARIETIES BY INDUCED MUTATIONS TO INCREASE YIELD PER ACRE AND RESISTANCE TO DISEASES AND TO IMPROVE SEED QUALITY*

A.J. MIAH, I.M. BHATTI, A. AWAN, G.BARI Atomic Energy Agricultural Research Centre, • Tandojam^ West Pakistan

« • Abstract

IMPROVEMENT OF RICE VARIETIES BY INDUCED MUTATIONS TO INCREASE YIELD PER ACRE AND RESISTANCE TO DISEASES AND TO IMPROVE SEED QUALITY. Mutagen treatments of the variety Kangni-27 have given rise to short culm and early ripening strains. Experiments have been started with the varieties Dokri Basmati, Jajai-77, IR-8 and 370 Basmati using fast neutrons, gamma rays, EMS and dES as mutagens. Results obtained so far show a differential response of the varieties in their radiosensitivity and frequency of leaf colour mutations.

INTRODUCTION

Since joining the FAO/IAEA Co-ordinated Program of Research on the Use of Induced Mutations in Rice Breeding in 1964 our work has been directed at improving rice by means of induced mutations. This paper gives the results of experiments that were- carried out from 1967 to 1968.

EXPERIMENT NO. I

This experiment was initiated in 1964; the details are given in a previous paper by Miah and Bhatti published In Rice Breeding with Induced Mutations, Technical Reports Series No. 86, page 75.

Short-culm and early flowering mutants in Kangni-27

(a) Short-culm mutants

Out of 21 short-culm mutants isolated in the M2 generation, 19 lines were found to breed true in the M3 generation. The plant progenies of these mutant lines were grown in a randomised block design with four replications in the M4 generation. Observations were made plant height at maturity ' and yield per plant. The results are presented in Table I. From the data it is evident that all the mutant lines are shorter than the original variety. The minimum average plant height was recorded in the mutant line S-8 (126.4 cm) which is 16.8% less than the original variety. The data also indicate that there is more variability in plant height character in the mutant

* This work was conducted under the IAEA Contract No. 290/RB.

69 70 MI AH et al. lines than in the mother line. Delay in harvesting due to shortage of labour resulted in high shattering of the grains and hence the yield data, though recorded, are not reliable.

(b) Early flowering mutants • •

Thirty-nine lines were selected from the M3 progenies of six early flowering mutants on the basis of earliness and higher plant yield. The plant progenies of these lines were raised in the M4 generation in a randomised block design with four replications1. The flowering date- and yield were recorded on every individual plant. The results are summarized in Table II. From the data it is seen that almost all lines flowered earlier than the original variety. It is also to be noticed that the variability for flowering » time is usually greater in the mutant lines than in the original variety. In •

TABLE I. PLANT HEIGHT AND YIELD OF 19 SHORT-CULM MUTANTS OF RICE COMPARED WITH THE ORIGINAL VARIETY KANGNI-27 IN

Plant height (cm) Plant yield Mutant lines (g) Mean Range

Original variety 151 .9 135.0 - 175.5 60.3

•S-3 134 •9 99.0 - 172.5 37.1

S-4 135 .0 96.5 • 167.5 42.7

S-5 133 .7 86.0 - 163.5 39.0

S-6 140 . 5 107.0 • 171.0 40.8

S-7 130. .4 97.5 • 166. 5 41.9

S-8 126 .4 •83.5 • 161.5 29.1

, S-9 134 .3 102.5 • 161.0 38.6

S-10 .. 135.. 5 106.5 • 170.5 .37.5

S-ll 138. .3 93.0 • 183.5 40.0

S-12 132. .6 102.5 • 169.0 38.5

S-13 132. .8 109.0 - 169.5 42.1

S-14 132. .8 78.0 • 167.0 40.2

S-15 ' 131, ,7 102.0 - 169.0 39.7

S--16 • . 134.. 9 92.0 - 170.0 41.5

S-17 • • • ' 134 .0 104.5 • 169.0 39.7

S-18< 135. .9 102.5 • 165.5 40.8

- S-19 . • • 134 .0 99'. 5 • 163.0 45.1 .

S-20 130 .4 91.5 • 167.5 37.5

S-21 129. .6 78.5 • 164. 5 '31.8 MIAH et al.

TABLE II. HEADING AND YIELD DATA OF 39 EARLY FLOWERING MUTANT LINES OF RICE COMPARED WITH THE ORIGINAL VARIETY'

KANGNI-27 IN THE M4 GENERATION

No. of days Days earlier ' Plant yield Mutant lines from sowing than original Range to heading variety

Original variety 98.7 96 - 104 39.1 EF-1-7 93.4 5.3 85 - 99 36.1 EF-1-47 97.4 J .3 92 - 100 39.1 EF-1-69 98.3' 0.4 .95 - 108 39.1 EF-1-112 97.7 1.0 95 - 100 41.8

EF-2-9 93.6 5.1 87 - 99 36.4 EF-2-51 94.1 4.6 85 - 98. 37.5 EF-2-73 94.5 4.2 88 - 98 35.1 EF-2-91 95.2 3.5 90 - 99 36.3

EF-3-2 95.2 3.5 89 - 99 40.9 EF-3-31 93.9 4.8 88 - 104 38.8 EF-3-68 93.0 '5.7 86 - 97 40.5 EF-3-86 94. 5 4.2 90 - 100 39.0

EF-4-18 93.1 5.6 79 - 97 31.3 EF-4-46 95.7 3.0 39.8 EF-4-57 95.3 • 3.4 89 - 99 29.2 EF-4-86 95.3 3.4 87 - 100 35.3

EF-5-4 91.9 6.8 85 - 9 8 29.1 EF-5-7 92.5 6.2 80 - 97 37.4 EF-5-9 91.0 7.7 85 - 95 32.5 EF-5-10 93.9 4.8 87 - 98 33.0 EF-5-11 89.8 8.9 85 - 96 30.8 ' EF-5-15 91.3 7.4 86 - 108 31.6 EF-5-23 90.3 8.4 82 98 27.8 EF-5-25 52.1 6.8 83 - 98 29.6 EF-5-27 9.6 83 - 95 29.2 EF-5-28 91.0 7.7 80 - 96 32.0 EF-5-30 90.0 8.7 83 -• 98 28.9 EF-5-50 90.0 8.7 79 - 97 31.2 EF-5-57 90.6 8.1 84 - ' 97 30.6 EF-5-58 91.1 7.6 80 - 97 29.7 EF-5-77 92.4 6.3 83 - 98 33.3 EF-5-82 90 2 8.5 83 - 95 33.6 EF-5-85 91 5 7.2 82 - 96 38.2 EF-5-100 90 6 8.1 83 - 107 32.6 EF-5-108 89.3 9.4 84 - 97 31.9

EF-6-1 97.2 1.5 94 - 104 22.7 EF-6-37 97.3 1.4 94 - 101 28.0 EF-6-67 96.6 2.1 95 - 103 28.2 EF-6-109 96.8 1.9 94 - 1011 28.4 72 MIAH et al. general, the yield per plant was reduced in the mutant lines but some lines had a yield potential at par.with the original variety.' This year 12 out of the 39 M4 lines have been put to micro-yield trial with four replications (plot size 1 5 ft X 6 ft).

EXPERIMENT NO.'II

Improvement in Dokri Basmati and Jajai-77

Resting seeds of the rice varieties Dokri Basmati and Jajai-77 were treated with 1000, 1300, 1500 and 1800 rads of fast neutrons by the IAEA laboratory at Seibersdorf, Austria, and with 20, 30, 40 and 50 kR of gamma radiation from the 60Co source at-the Atomic Energy Agricultural Research Centre, Tandojam at a dose-rate of 8.5 kR/h. The Mx generation was grown in summer 1967 and observations were made on germination, plant height at maturity and seed fertility.

For chlorophyll mutation studies in the M2 generation, the longest spike from each Mj plant was sown, without threshing, as an M2 strain. Chloro- phyll mutants were scored when the seedlings were about 14 days old. Strains with less than 10 M2 seedlings were discarded while arranging experimental results. Mutation frequency was expressed as the number of mutations per 100 Mj spikes and also as the number of mutants per 1000 M2 seedlings. The segregation ratio for each treatment was a pooled one, i. e. expressed as a percentage of total mutants in total tested M2 seedlings in mutated M2 strains. Chlorophyll mutations were classified into four groups, i.e. albina (white), xantha (yellow), viridis (light green) and others (no definite pattern of chlorophyll destruction). Chlorophyll mutation studies were carried out in a field nursery from May to July, 1968.

M2 plants have been grown in the field for studies of viable mutations of economic importance. The results of the Mj generation studies are presented in Table III. The results show that germination, plant height and seed fertility of the variety Dokri Basmati decreased after neutron and gamma irradiations, but the decrease was not proportional to the increase in dosage. In Jajai-77, germination, plant height and seed fertility of the treated populations showed the same tendency as in Dokri Basmati, except for germination after gamma-ray treatments, which increased above the non- irradiated control. From the data it is apparent that Jajai-77 is more radioresistant than Dokri Basmati. Results of the chlorophyll mutation studies are presented in Table IV. In Jajai-77, both the number of mutations per 100 Mj spikes and the number of mutants per 1000 M2 seedlings, after neutron irradiation, increased with dose, but in the highest dose (1800 R), the mutation frequency dropped remarkably. On the other hand, after gamma irradiation the mutations per 100 Mi spikes decreased with the increase in dose. Mutant frequency per 1000 M2 seedlings increased up to 30 kR and then decreased at higher doses. In the variety Dokri Basmati, mutation frequency on the basis of Mj spikes and mutant frequency per M2 seedlings were found to be highest after neutron irradiation with 1800 R, but at 1300 and 1500 R the mutation VIADO et al. 73

TABLE III. DATA ON GERMINATION, PLANT HEIGHT AT MATURITY AND SEED FERTILITY OF Mx PLANTS OF RICE'VARIETIES DOKRI BASMATI AND JAJAI-77 AFTER IRRADIATION

Germination Plant height Seed fertility Variety Treatment (% of control) (6/o of control) of control)

Fast neutrons (R)

1000 77.0 96.6 58.0 1300 84.7 92.3 42.5 ' 1500 80.9 93.1 .43.5 • 1800 82.5 ' 90.4 36.5' Dokri Basmati Gamma rays (kR)

20 95.7 91.8 74.0 30 98.4 88.8 59.4 40 89.7 87.2 48.1 50 .94.6 90.7 39.6

Fast neutrons (K)

1000 94.3 98.2 71". 3 1300 105.7 97.5 52.3 • 1500 110.3 95.7 49.2 1800 104.6 95.8 58.3 Jajai-77 Gamma rays (kR)

20 97.3 98.1 75.2 30 95.7 96.5 57.9 40 97.8 93.5 55.0 50 82.3 . -97.9 45.0

frequency was lower than at 1000 R. After gamma-irradiation, the mutation rate (calculated per 100 Mj spikes and per 1000 M2 seedlings) increased up to 30 kR and then decreased at higher doses. The data also indicate that neutron irradiation induced a higher frequency of chlorophyll mutations than gamma irradiation in both varieties. In both varieties, no linear increase • in segregation ratio could be observed after neutron or gamma-ray treatments. The segregation ratio showed almost the same tendency as the mutation frequency per 1000 M2 seedlings. The mutation spectra obtained in different treatments with neutrons or gamma rays were pooled together and the results are reported in Table V. The data show that in both Dokri Basmati and Jajai-77, the appearance of albino mutations was maximum after neutron irradiation, followed by viridis in the case of Dokri Basmati and by xantha in the case of Jajai-77. On the other hand, after gamma irradiation, viridis was followed by albina in both the varieties. TABLE IV. . CHLOROPHYLL MUTATION FREQUENCY IN M2 IN RICE VARIETIES DOKRI BASMATI AND JAJAI-77

Mean Average M2 Mi spikes Mutations per 100 Mg seedlings Mutants per 1000 Variety Treatment segregation ratio seedlings analysed Mi spikes analysed Mj seedlings (%) per spike

Control 119 5468 - - 46.0

Fast neutrons (R)

1000 104 16 .4 3468 21.9 15.4 33.4 1300 86 8 .1 2791 9.2 8.4 32.5 15 00 88 17.. 0 2474 16.2 11.2 28.1 1800 .76 26,. 3 2087 34.5 13.6 27. 5 Dokri Basmati

Gamma rays (kR)

20 49 22., 5 1847 8.1 3.65 37.7 30 49 30 .6 1607 26.8 8.8 32.8 40 46 15 .2 1260 17.5 11.4 27.4 50 69 23,. 2 1608 24.9 10.7 23.3

Control 113 11.. 5 9848 1.4 1.2 87.2

Fast neutrons (R)

1000 66 21, .2 4561 6.8 2.7 69.1 1300 98 32, ,7 5494 25.3 7.9 56.1 1500 88 33. ,0 5428 50.5 12.8 61.7 1800 98 19. 4 6098 12.0 7.1 62.2 Jajai-77 Gamma rays(kR)

20 93 35. 5 6362 14.0 3.7 68.4 30 80 20. 0 4165 . 18.3 7.4 52.1 40 85 16. 5 3998 17.0 9.4 47.0 50 73 15. 1 3336 11.4 10.4 45.7 VIADO et al. 75

TABLE V. SPECTRUM OF CHLOROPHYLL MUTATIONS (VALUES IN%) IN RICE VARIETIES DOKRI BASMATI AND JAJAI-77 AFTER NEUTRON AND GAMMA IRRADIATION

Total No. Variety Mutagen Albina Xantha Viridis Others of mutations

Fast neutrons 63.5 12.8 15.6 8.1 211 Dokri Basmati Gamma rays 31.7 8.3 41.7 18.3 120

Fast neutrons 60.7 19.0 9.7 10.6 517 Jajai-77 Gamma rays 37.3 17.0 38.0 7.7 271

TABLE VI. GERMINATION AND PLANT HEIGHT AT MATURITY OF Mj PLANTS OF RICE VARIETY IR-8 AFTER GAMMA IRRADIATION

Dose Germination Plant height (cm) as (kK) (

20 128.4 97.5

30 111.9 96.7

40 111.0 95.9

50 105.7 93.3

EXPERIMENT NO. Ill

Improvement in IR-8 variety

Four doses of gamma radiations, i.e. 20, 30, 40 and 50 kR, were given to dry seeds of the rice variety IR-8 at the Research Centre, Tandojam. In the Mj generation grown in the summer of 1967 germination and plant height characters were studied. The data are presented in Table VI. From the data it is evident that germination increased in all the treated populations compared with the control. The increase was highest at 20 kR and then decreased with the increase in dose. The plant height, on the other hand, decreased with the increase in'dose. The M2 generation was raised in 1968 for the study of viable mutations of economic importance. 76 VIADO et al.

EXPERIMENT NO. IV

Improvement in 370 Basmati and IR-8

Dormant seeds of two rice varieties, namely 370 Basmati (fine grain) and IR-8 (coarse grain) were subjected to the following mutagenic treatments in May, 1968.

(a) Gamma-ray treatment

A60Co gamma source of the Atomic Energy Agricultural Research Centre, Tandojam was used at a dose-rate of 5.4 kR/h.

(b) Chemical treatment

Fresh solutions of EMS and dES were prepared with deionized water. After a 3-h presoaking, the seeds were treated with the chemicals for 20 h at room temperature and then washed thoroughly. Presoaking and post- treatment washing were also done with deionized water.

(c.) Doses given

Mutagen - Dose Gamma rays 25, 35 and 45 kR EMS ' 0. 1, 0. 3 and 0. 5% dES 0. 1, 0.15 and 0.2%

(d) Handling after treatment.

After the seeds had been treated with the mutagens they were sown in the nursery and the seedlings were transplanted in the field after 30 days of sowing. Seed fertility and morphological variations, if any, will be studied in the Mj generation.

DISCUSSION

N. YAMADA: I wonder if one can expect a higher yield by reducing the plant height of any variety. If not, what is the criterion for selecting the mother variety for mutagen treatment? A.J. MIAH: I think it is usually possible to increase the yield by reducing the plant height. In that case the starting material should be tall because if the starting material is already very short.there is very little possibility to increase the yield by further reducing the plant height. On the other hand, if the objective is to increase the yield without decreasing the plant height, the starting material should be of good quality or have some other desirable character. And the increase in yield in that particular material should be aimed at by inducing polygenic'mutations in the yield components. BREEDING FOR EARLINESS, HIGH YIELD AND DISEASE RESISTANCE IN RICE BY MEANS OF INDUCED MUTATIONS

M.S. HAQ, S.M. ALI, A.F.M. MANIRUZZAMAN, A. MANSUR and R. ISLAM Atomic Energy Centre, Dacca, East Pakistan

Abstract

BREEDING FOR EARLINESS, HIGH YIELD AND DISEASE RESISTANCE IN RICE BY MEANS OF INDUCED MUTATIONS. Ten varieties of Boro, Aus and Aman rice were treated with 30 kR of gamma rays from the 500-Ci 60Co source of the Atomic Energy Centre of Dacca. In addition, two rice varieties were treated with EMS and dES. '

To suppress tillering, the seeds were sown late and at a high seed rate. 300 normal-looking fertile Mt

plants from each variety were harvested at random. The M2 progenies were sown on a plant-to-row basis to select for high yield, earliness, blast resistance and response to large doses of nitrogen fertilizer. Characters like plant height, number of tillers, fertile tillers, length of panicle and time from sowing to maturity were checked. Asthe results showed great variability in the mutagen-treated material the chances for successful selection are promising.

INTRODUCTION

The present mutation breeding work was initiated in December 1966 with the object of evolving mutants of rice which are early'and high yielding, resistant to blast disease, and responsive to heavy fertilization without being lodged by using a physical mutagen (gamma rays) and chemical mutagens (EMS and dES). The importance of such breeding programs needs no emphasis as the local varieties, which are all indica type, are lacking in most of the above qualities. The per acre yield of rice is very low (1500 lb), the plants are tall with weak straw, incapable of utilizing more fertilizers and have the tendency to lodge. Earliness is a very desirable character for East Pakistan conditions as it will ensure a safe harvest before the floods and will help in growing more crops in one year in the same field.

MATERIAL AND METHODS

In East Pakistan three different groups of rice are grown in three different rice growing seasons: Boro (November-December to April-May), Aus (April-May to July-August) and Aman (July-August to December- January). Two varieties of Boro rice (Habiganj-II and Habiganj-VI), four varieties of Aus rice (Kataktara, Marichbati, Dular and Dharial) and four varieties of Aman rice (Nizersail, Latisail, Chitraj and DA-31) have been used in this program.

77 78 VIADO et al.

From both laboratory and field experiments 30 kR of gamma rays was found to be the most convenient working dose for all these varieties. Irradiation was done from a 500-Ci 60Co gamma source of the Atomic Energy Centre, Dacca. Seed samples were moisture stabilized (12-14%) before irradiation.

M] generation

The Boro seeds were sown broadcast and late at a higher seed-rate of 125 lb/acre instead of the usual rate of 82 lb/acre in order to suppress tillering. The usual practice with Boro is to sow the seeds in the nursery bed in early November and transplant the seedlings in the field in early December. The Aus varieties were sown broadcast (which is the usual practice), late and with a higher seed rate for the same reasons. The Aman rice varieties were first sown in the nursery bed in late August and transplanted in the field in the later part of September, also to suppress tillering. The size of the population was approximately 100 000 plants for each of the varieties under each group of rice. At maturity, 300 normal-looking fertile plants were selected at random from each variety of Boro, Aus and Aman. Data relating to plant height, tiller number, number of panicles, and sterility percentage were recorded at maturity for each plant. The seeds of the selected plants were kept separately. The seeds of the rest of the plant population were bulked.

M2 generation

In the second year of the experiment the 300 individual selected plants were divided into three lots of 100 each, and the seeds from each of the plants were grown on a plant-to-row basis. The three groups of 100 plants were for the following three experiments:

1. Selection of early and high-yielding mutants; 2. Selection of blast-resistant mutants; and 3. Selection of mutants responsive to large doses of nitrogenous fertilizer.

In addition, the M2 bulk population from the bulk Mj^ seed were grown for further selection work.

1. Selection of early and high-yielding mutants (Aus-, Aman and Boro)

The seeds from 100 selected Mi single plants were sown well spaced in 100 rows of 10 plants each in each replication, there being two replications. The following observations on a single plant basis were recorded.

(a) Date of sowing (b) Date of flowering (c) Plant height (d) No. of tillers (e) No. of fertile tillers. VIADO et al. 79

2. Selection of blast-resistant mutants (Aus and Aman only)

For selection of blast-resistant mutants (Aus and Aman), seeds from 100 selected Mi single plants were exposed to infection by the uniform blast nursery trials' technique recommended by the IRC and IRRI. Boro seeds were not exposed to blast infection. It has been our observation that the incidence of blast in Boro rice is of minor importance. The Boro season is characterized by relatively dry weather, short days and low temperature.

3. Selection of mutants responsive to large doses of nitrogenous fertilizers (Aus, Aman and Boro)

The 100 selected Mi single plants from each variety were planted well spaced in 100 rows of 20 plants each. The land was fertilized at the rate of 120 lb N2 (urea), 60 lb P2 Os (T.S.P.) and 60 lb K20 (muriat of potash) per acre. The plants were harvested on a single plant basis.

Treatment with chemical mutagens: (Mi)

Work was started with two rice varieties (Nizersail and Latisail Aman varieties) from late 1967. The treatment of pre-soaked seeds (soaking time 24 h) with freshly prepared mutagen solutions, which were prepared with de-mineralized water, was carried out at pH 7. 0 at a room temperature of 30°C. The pH was maintained with a buffer. The dES treatment time was 2 h for a 0.03M solution and the EMS treatment time was 24 h for a 0.03IVI solution. Large volumes of mutagen solution (1 ml/seed) were used and the solution containing the seeds was vigorously shaken by a mechanical shaker to give each seed the opportunity to absorb the same number of moles of mutagen. Post-treatment washing was done in a buffer solution for about 1 h. With d'ES, the concentration was kept relatively constant by changing to fresh solutions after one fourth had been hydrolyzed. Sowings were made after post-treatment.

RESULTS AND DISCUSSION

M1 generation

Table I shows the Mj data for different characters of different varieties under Boro, Aus and Aman groups of rice. It may be mentioned that the Boro crop was raised out of season and sown broadcast. The Aus and Aman crops were sown late. In all cases the seed rate was higher' than the normal. These untimely, late and thick sowings seemed to be very effective in lowering the tiller numbers.

Chemical mutagens (Mj)

All the plants of the two varieties were harvested on a single plant basis for growing during normal time in the M2 generation. 80 HAQ et.al.

TABLE I. AVERAGE NUMBER OF TILLERS PER PLANT. OF CONTROL AND IRRADIATED Mj RICE VARIETIES, 1966 - 1967

Tiller No. per Tiller No. .plant sown at "Jo suppression Rice group Variety per plant normal time in tillering (av.) (av.)

Boro Habiganj-II 3.1 10 - Control

1966-67 Habiganj-II 3.9 - 61 Irradiated

Habiganj-VI 4.3 12 - Control Habiganj-VI 4.5 _ 62.5 Irradiated

Aus Kataktara Control 2.6 8 1967 Kataktara ; Irradiated •3.5 - 56.3

Marichbati

Control 1.7 • ' 6 -

Marichbati

Irradiated 2.2 - 63.3

Dharial

Control 1.9 8 -

Dharial •

Irradiated 2.4 - 70.0

Dular

Control 2.9 6 -

Dular

Irradiated 3.4 - • 43.3

Aman Nizersail

Control 8.5 12.3 -

1967-68 Nizersail

Irradiated 6.6 - 46.3

Latisail Control 7.7 11.6

Latisail

Irradiated 7.0 - 39.6

Chitraj

Control 6.2 " 8.4 -

Chitraj

Irradiated 5.4 ' ' - 35.7

DA-31

Control 4.7 6.7 -

DA-31

Irradiated 4.4 • - 34.3 VIADO et al. 81

M2 generation

The M2 generation of Boro was grown during'Nov. 1967 to May 1968. The M2 Aus crop is in the field awaiting harvest and the M2 Aman crop will be sown next August. Table II shows the range of variation in the different characters. The M2 results for Boro show a great variation in the mutant lines compared with the control (see Table II and Appendix.I). Marked variations are observed in the characters of plant height and number of total and fertile tillers. ' There are a few lines and plants which are comparatively short in height. If these lines and plants breed true'in the M3 generation they may be of value to us as these may be resistant to lodging and more responsive to nitrogenous fertilization. As a result of circumstances beyond our control the experiment with Habiganj-VI (Boro) was abandoned.

TABLE II. RANGE OF DIFFERENT CHARACTERS FOR CONTROL AND IRRADIATED M2 HABIGANJ-II BORO VARIETY, 1967 - 1968

Length of Seeding to Plant height Total No. Fertile Habiganj-II(Boro) panicle maturity (cm) of tillers tillers (cm) (days)

Irradiated 68 - 123 5/5-48.0 3.5-43:0 18-27 144-160 (mean of lines)

Control (mean) 118.5 16 16 22.4 144

ACKNOWLEDGEMENTS

The financial and technical assistance of FAO and IAEA is gratefully acknowledged. Thanks are due to all the staff members of the Plant Breeding and Genetics Section of the Agriculture Division of Atomic Energy Centre, Dacca, for their co-operation and assistance. The visit to the experimental plots and the discussion of the problems with the investigators by Dr. Lars Fredriksson, Uppsala, Sweden, and Dr. BjOrn SigurbjCrnsson, Joint FAO/IAEA Division of Atomic-Energy in Food and Agriculture are greatly appreciated. 82 VIADO et al.

APPENDIX I

HABIGANJ-II BORO MUTANT LINES Date of sowing 14.11.1967

Mutant Plant _ t , „ ., Panicle u • Total Fertile , . .. t , line height ..„ length Date of flowering , ° tiller tiller , ° No. (cm) (cm)

1 120 22.5 21.5 21.3 8.4.68

2 . 116 23.0 11.0 26.2 9.4.68

3 112 37.5 30.0 18.0 10.4.68

4 109 25.2 16.4 22.5 7.4.68

5 104 7.0 4.0 22.9 3.4.68

6 118 18.0 17.0 25.7 7.4.68

7 112 14.2 12.7 23.6 7.4.68

8 .103 18.0 18.0 19.5 9.4.68

9 • 111 33.0 32.0 21.5 3.4.68

10 112 24.3 21.6 22.2 9.4.68

11 114 20.0 18.0 23.4 10.4.68

12 112 16.0 9.0 23.3 31.3.68

13 119 17.6 16.9 23.3 2.4.68

14 117 16.4 14.0 21.4 4.4.68

15 118 17.6 16.6 22.3 2.4.68

16 117 19.0 17.0 21.6 1.4.68

17 114 21.0 19.0 22.0 1.4.68

18 104 36.0 36.0 21.0 1.4.68

19 111 24.0 23.0 22.0 1.4.68

20 119 25.0 21.0 22.7 1.4.68

21 115 21.0 12.5 21.7 1.4.68

22 68 16.0 15.3 23.6 1.4.68

23 105 13.0 12.6 21.6 2.4.68

24 94 8.0 6.0 22.6 3.4.68

25 108 16.0 15.6 19.5 3.4.68

26 100 14.0 13.3 20.3 2.4.68

27 96 18.0 18.0 22.7 5.4.68

28 95 20.2 19.0 21.3 3.4.68

29 91 20.0 18.0 19.7 4.4.68

30 105 16.5 14.0 22.8 2.4.68 VIADO et al. 83

Appendix I (cont. )

Mutant Plant Panicle Total Fertile line height length Date of flowering tiller tiller No. (cm) (cm)

31 110 19.0 13 .0 21.1 4.4.68

32 97 16.0 6 .0 19.0 1.4.68

33 101 9.5 8.. 0 21.0 5.4.68

34 114 22.3 22 .0 21.5 1.4.68

35 115 22.0 18 .0 21.3 7.4.68

36 113 26.0 24.. 0 21.5 4.4.68

37 123 35.5 30.. 0 26.4 1.4.68

38 111 35.0 35.. 0 22.2 12.4.68

39 110 24.5 23 .5 22.7 25.3.68-

40 106 48.0 43 .0 20 i 0 7.4.68

41 103 32.5 30,, 5 23.7 9.4.68

42 109 22.0 18.0 25.3 6.4.68

43 112 20.5 17 .5 • 23,. 7 19.3.68

44 118 28.0 24,. 0 21.5 6.4.68

45 111 20.0 , 17.. 5 23.5 2.4.68

46 83 5.5 3,, 5 19.8 2.4.68

47 104 16.0 16., 0 21.8 26.3.68

48 117 32.0 32,. 0 22.0 8.4.68

49 108 22.5 20. .5 21.6 27.3.68

50 93 32.0 31. ,0 27.0 3.4.68

51 111 26.0 21. 0 25.5 26.3.68

52 103 15.0 10,, 0 22.5 20.3.68

53 110 27.5 C O C O ,0 23.0 3.4.68

54 102 13.0 9., 0 23.2 26.3.68

55 114 35.0 34. ,0 20.0 10.4.68

56 102 23.0 20. 0 23.5 1.4.68

57 107 28.0 21. .5 22.5 20.3.68

58 108 24.0 21. 0 23.3 20.3.68

59 101 25.0 23. 5 21.9 30.3.68

60 103 27.0 8. 0 22.0

INDUCTION AND UTILIZATION OF MUTATIONS IN RICE *

G.B. VIADO1, I.S. SANTOS2, E. CADA3, P.B. ESCURO4, J.D. SORIANO5

Abstract

INDUCTION AND UTILIZATION OF MUTATIONS IN RICE. Experiments at the Philippine Atomic Research Center with gamma-irradiated seeds of IR-8-2 88-3 showed a pronounced inhibition of germination at 60 and 70 kR. The LD50 was calculated to be about 54 kR at 8 days from seeding. Fertility decreased with increase in gamma radiation dose in an approximately linear'fashion. Numerous types of variants have been obtained in the M2 generation of which the' following may. be of particular interest: (1) Plant height slightly shorter and leaves darker green, narrower and more erect than IR-8; 99 days from seeding to harvest, fertile. (2) Plant height distinctly shorter than IR-8, leaves dark green small and stiff, culms small, produced as many as 27 panicle-bearing culms, grains very narrow. (3) Typically IR-8 in early growth but at maturity exhibits long panicle exertion, longer grains than IR-8, 111 days from seeding to harvest, produced as many as 20 productive tillers.

At theU.P. College of Agriculture, Los Banos 308 plants were'selected from the M 2 generation of

BE-3 and BPI-121, and 85 from the M'3 generation of Ben ga wan, Peta and In tan after irradiation with fast neutrons. All these selections were early to medium-early and short to medium-short. More beneficial mutants were obtained with the.lower dose treatments of BE-3, BPI-121 and Bengawan.and with the higher dose treatments of Peta. Plant-progeny rows of these selections, except those from the photo - period-sensitive varieties BE-3 and BPI-121, are being grown this 1968 dry season for further evaluation.

In the M4 generation of Peta a larger number of short.to medium-short and early to medium-early plants were selected from the 35 to 45 kR gamma treatments and the 0.5 to 1.0% EMS treatments. A total of 2430 plant selections were harvested and are now being grown in plant-progeny rows. Seventy-three uniform lines were selected from the M 3 generation of Peta and Intan irradiated with fast neutrons and from the M4 generation of Peta treated with gamma rays, mixed radiations and EMS. All these lines were earlier and mostly shorter than the Peta check. .They are now growing in a twOT replicate preliminary test with.the control. . , • Work at the Maligaya Rice Research and Training Center has included (1) further selection of mutants in the M3 of several- varieties after different mutagenic treatments, and (2) field tests and observations on 12 indica and 15 japonica mutants. From BPI-121, 140 non-seasonal lines .were selected. The range of maturity of the selected lines was 120 - 125 days from sowing to harvesting; From BE-3, 547 lines were selected for non-seasonal characteristics with a-range of maturity from 125 to 140 days. Of these selections 12 from BPI-121 and 28 from BE-3 were practically uniform and are now being tested for preliminary yield trials during the 1968 dry season. In the field trials four of the indica mutants gave higher yields than the check. All the indica mutants were non-lodging and relatively early. The japonica mutants did not perform as well as the indica mutants. All were non-lodging but only the irradiated Taichung 65 Mut. gave a higher yield than BPI-76 (check).

* Supported by FAO/IAEA research contract No. 28. 1 Philippine Atomic Energy Commission, . Manila, Philippines 2 Philippine Atomic Research Center, Dili man, Quezon City, Philippines 3 Maligaya Rice Research and Training Center, Munoz, Nueva Ecija, Philippines 4 U. P. College of Agriculture, College, Laguna, Philippines 5 University of the Philippines, Diliman, Quezon City, Philippines

85 86 VIADO et al.

A study on the-selection for eating quality in M2 IR-8 rice at the College of Arts and Sciences, University of the Philippines indicated that improved eating quality may be induced in IR-8 by mutagenic agents.

I. MUTATION EXPERIMENTS AT TI-IE PHILIPPINE ATOMIC RESEARCH CENTER, DILIMAN, QUEZON CITY

A. STUDIES ON RADIOSENSITIVITY

1. Material and methods

F7 seeds of IR-8-288-3 (IRRI), with a moisture content of 11.9%, were given acute exposure in air to 10, 20, 30, 40, 50, 60 and 70 kR of gamma radiation from the 60Co gamma irradiation facility of the Philippine Atomic Research Center. Two-hundred seeds were used in each treatment. For the irradiation each lot of seeds was divided and placed in two uniform layers on two pieces of cellophane tape, 1. in. X 3 in. each. The irradiated seeds were soaked in distilled water for two hours before they were sown in seed boxes containing garden soil. In all the plantings, each treatment was replicated four times. Only 10 random plants from each replication were used for data in Tables I and II. In the seedling stage of observations were made on:

The per cent germination and per cent abortive germination; The seedling height at 8 days from seeding; The number of seedlings showing morphological abnormalities; The per cent survival to 21 days (transplanting); and

The LD5o (at 8 days from seeding).

TABLE I. RADIATION EFFECTS ON THE GROWTH OF IR-8-288-3 SEEDLINGS FROM GAMMA-IRRADIATED SEEDS Based on 40 plants per dose

Germination „ . , - , Survival . , . Seedling height , > morphologically Dose Total germination 7° abortive germination ° ° to 21 days , r ,,. ° (> control) . , defective seedlings (kR) (°/o control) 07° control) 5

0 100 0 100 100 4.0

10 98.9 0.5 100 100.2 22.8

20 97.3 2.1 91.7 100.7 20.5

30 100.5 0 85.4 89.8 33.5'

40 96.3 7.0 50.0 78.7 51.0

50 91.4 28.0 50.0 61.1 86.5

60 71.1 78.0 33.3 24.5 98.2

70 50.3 100 - - - VIADO et al. 87

TABLE II. EFFECT OF GAMMA RADIATION ON Mj PLANTS OF IR-8-288-3 Based on 40 plants per dose

Average No. Average No. Average No. Average Average Dose of panicle-bearing of days from sowing of branches per hill pollen fertility seed set (kR) culms per hill to heading

0 14.2 0.3 97.6 91.0 76.5

10 16.5 1.0 96.8 77.6 64.1

20 17.0 7.0 95.8 70.2 46.7

30 15.8 8.2 97.8 50.8 42.8

40 17.2 12.3 98.6 • '31.4 33.5

50 14.4 13.2 98.8 20.3 18.0 b 60 a a a 9.6

a Very few plants, k Very few seeds formed.

In the mature stage of M^, observations were made on:

The per cent pollen fertility according to the iodine test; The per cent seed set based on the first three panicles that emerged; The number of panicle-bearing culms per hill; and The average number of days from sowing to harvesting.

2. Observations 011M1 seedlings

Data taken during the seedling stage are summarized in Table I. The data show that the early stage of germination was not inhibited by doses up to 40 kR. At 50 kR, inhibition is perceptible, and at 60 and 70 kR it is pronounced. The completion of germination appeared to-be severely inhibited at 50 kR and higher doses. On the basis of seedling height, Table I shows that IR-8-288-3 tolerates gamma radiation without sustaining severe reduction up to 30 kR. Morphologically distorted and defective seedlings were observed even in the lowest dose level used (10 kR) and their number markedly increased with increase in dose. Survival of the seedlings to 21 days C (up to the time of transplanting) was reasonably high except at 60 and 70 kR. The LDg0 was calculated to be approximately 54 kR at 8 days from seeding.

3. Observations on mature Mj plants.

There is a slight indication that the radiation treatment might have caused a certain increase in the number of panicle-bearing culms. Another effect observed at this stage of growth was the production of 88 VIADO et al. secondary branches at the nodes of many culms which were also • panicle bearing. A count of the number of branches produced per hill revealed that this branching effect increases with increase in dose level (Table II). The number of days from seeding to emergence of the inflorescence did not appear to.be affected by the radiation treatment. However, both the percentage of fertile pollen and the percentage of filled grains decreased with increase in gamma;radiation dose in an approximately linear-fashion (Table II).

B. MUTANTS IN THE M2 GENERATION

From each treatment dose 4 X 10 plants were harvested (first 3 panicles per plant). The seeds of the 40 lines were bulked and a sample of about 2400 seeds from each treatment was planted in June 1.967 for M2 observation. Heavy rat infestation, however, reduced the number of plants. The plants were checked for the following characters: (a) At seedling stage: "Colour (b) At growth stage: ' . Relative height Colour, shape and size of leaves Degree of tillering and variations in the culms Days from seeding to heading (c) At maturity:' " • ' ' - 1 Number of productive tillers Fertility ' ' 1 Panicle characteristics ' Grain characteristics.

M2 observations have not yet been completed. Only partial results from M2 plantings can be reported at pres:ent, and the descriptions, can be regarded as tentative until checked and confirmed in the M3 and compared with those reported in the literature. The characteristics of'typical IR-8-288-3 are: high tillering ability, short stature (90 - 105 cm), moderately early maturity (120 days from ' seeding to harvest), insensitivity to photoperiod, 'susceptible to bacterial leaf blight, highly susceptible to certain races of rice blast prevalent in the Philippines, medium-sized and chalky grain. ;

Types of variants observed in the M2 • 1 ' ' . '

Besides the albino, xantha and.mottled leaf seedlings, the", following types of variants have been observed:.-" ......

1. Leaf wide and more erect than typical IR-8, blunt leaf apex, base of plant very compact, culm stout, grains rounded. 2. Loose plant habit, leaves light green, 'le'af blade narrow and folded at midrib, panicles long, grains long and very narrow with very strong attachment, 139 days from seeding to harvest. 3. Very compact plant habit,' 104'days from' seeding to harvest, produce as many'as 41'panicle-bearing, culms; semi-sterile.' VIADO et al. ,89

4. IR-8 plant habit, 173 days from seeding to harvest (probably photoperiod sensitive), dense panicle,' large peduncles, tendency to shatter. : • >. . 5. Very short plant, leaves onion-like (fine and-rolled), culms very, small, grains smaller. • . . •' 6. Leaves narrower and longer than typical IR-8,- taller with long internodes,. duration as long as 172 .days from seeding to harvest (probably photoperiod sensitive),- glumes streaked with brownish, pigmentation. : i 7. Slightly shorter than IR-8, dark green, leaves narrower and more erect, 99 days from seeding to harvest, fertile. 8. Plant habit that of IR-8, tillers with chimeral yellow variegation. - 9. Similar to (8) but white variegation. 10. Plant habit like IR-8 but the colour of,the leaves distinctly yellow green.. • . . . 11. Plant habit like IR-8, grains much smaller in length and width. 12; Distinctly shorter than IR-8, leaves smaller and more erect, culms also small. • • 13. Distinctly shorter than IR-8, leaves dark green, -small and stiff, • culms small, produce, as many as 27. panicle-bearing culms, grains very narrow. 14. Extremely small (30 cm height), grass-like in appearance. 15. Shorter than control, .leaves somewhat folded, grains long. 16. Leaves longer and narrower than .control, very tall, lodging, bushy habit, culms branching at the nodes, branches also panicle-bearing, 172 days from seeding to harvest of main culms (probably photoperiod sensitive). 17.. Typically. IR-8 in early growth, but a mature stage plant exhibits long panicle exertion, longer grains than IR-8, 111, days from seeding to harvest, produces as many as 20 productive tillers. 18. Early growth is indistinguishable from IR-8, at transition to flowering stage plant habit becomes spreading, panicle lax with long .exertion., grains very long, 111 days from seeding to harvest. 19. Morphologically similar to IR-8, except for narrower leaves, grains' •very long, 111 days to harvest. 20. Early growth habit similar to IR-8, beak or claw-shaped grains, opaque kernels, 111 days to harvest, semi-sterile-. 21. Morphologically similar to control but grains; are rounded. 22., Leaves narrower than control, folded or cupped, semi-sterile. 23.- Plant habit-indistinguishable from control but 105 days to harvest, semi-sterile. , " - - 24. Leaves distinctly narrow, base of plant very compact, purplish in colour,; tillers many, panicles do not fully emerge, spikelet possesses only one functional glume the other being modified into a long awn-, like structure, kernel is exposed, very low seed set. • ' 25. Shorter than control, leaves also.smaller, semi-sterile. 26. Relatively tall with long internodes, grain with.purple'apiculus; very low fertility. '. ; . i . • 27. Morphologically normal, semi-sterile (probably approximately 50% seed set). 28.- Morphologically.normal, very low.fertility (seed set probably only 5%). 29. Morphologically normal but completely sterile. . 90 VIADO et al.

30. Non-tillering, usually sterile. 31. Small, with small leaves and culms, sterile. 32. Leaves lighter green than control, narrower and longer, late heading, panicles do not emerge completely, no grains. 33. Small stiff plants, leaf blade has prominent veins, few tillers, no panicle emerged after booting, probably sterile. 34. Leaves long, loose growth habit, later crawls in grass-like manner producing shoots and roots at nodes, low fertility. 35. Loose plant habit, no uniformity in growth, 219 days to maturity.

C. SELECTION IN LATER GENERATIONS

Seeds of variants observed in the M2 were planted for the purpose of checking the heritability of the variations. The plants are at present in the seedling stage. About 3300 mutant lines were sent to the U.P. College of Agriculture, College, Laguna, Philippines, and are at present being screened for blast resistance. More plant lines will be tested for blast disease resistance as soon as more seed material can be processed.

II. EXPERIMENTS AT THE U.P. COLLEGE OF AGRICULTURE, COLLEGE, LAGUNA

1. Description of work

(a) Cultivation of M2 and M3 generations of lowland rice seeds irradiated with fast neutrons

M2 seeds harvested from the 1966 wet-season planting of treated seeds of rice varieties Bengawan, Peta and Intan (weakly photoperiod- sensitive varieties) were sown in an ordinary wet bed on January 9, 1967. The seedlings were transplanted singly in hills in 6-m plant-progeny rows in the lowland field 18 days after sowing. The hills were 30 cm X 20 cm apart. Ten rows of progenies of untreated seeds were also grown beside the M2 plants of each variety. No fertilizer was applied in the experimental field because of the high native fertility of the soil as a result of previous fertilization. During the past rainy season, even without fertilizer, the local varieties lodged badly. Insect pests like stem borers and leaf hoppers were controlled with two applications of gamma BHC granules at 3 kg a.i. per ha and bi- weekly spraying of "Sevin" insecticide at 2 kg a.i. per ha within two months from transplanting. The weeds were adequately controlled by rotary weeding followed by hand weeding. At maturity, early and short-statured plants were selected. Plants and rows with characters similar to those of the control were discarded. Sixty- five plant selections from Bengawan, 93 from Peta and 22 from Intan were harvested.

During the following rainy season, the M3 generation of these selections was sown in an ordinary wet bed on July 8, 1967, and transplanted singly in hills in 2.75-m progeny rows on August 1. The hills were VIADO et al. 91

30 cm X 25 cm apart. Progenies of untreated seeds of each variety were grown after every 20*h row. The M2 generation of three varieties (BPI-121, BE-3 and Raminad Strain 3), which were not grown during the preceding dry season because of their photoperiod sensitivity, were sown in an ordinary wet bed on July 9 and were transplanted in the same manner as the M3 materials using 6-m rows. • Recommended cultural practices were followed. Grass weeds were controlled by spraying them with "Treflan" herbicide at 1 kg a.i. per ha within a week after transplanting followed by a 2, 4-D spray about three weeks later to control broad-leaf weeds. Remaining weeds were hand pulled. Stem borers'were controlled with Diazinon granules at 3 kg a.i. per ha applied at monthly intervals. When leaf hoppers became abundant in neighbouring areas later in the season, the field was sprayed with Sevin insecticide at 2 kg a.i. per ha.

(b) Cultivation of M3 and M4 generations of mutagen-treated Peta and Intan

Seeds of 526 M2 plant selections were sown as lines in an ordinary wet bed on January 8, 1967. Most of these (453 lines) were selections from progenies of EMS-treated Peta. Twenty days later the seedlings were transplanted singly at 30 cm X 20 cm spacing in plant-progeny rows, 6 m long. Recommended cultural practices, similar to those followed for the M2 fast-neutron treated plants stated above, were followed. Plant selections were practised as the plants matured. Those selected within the first week were considered early and those selected within the second and third weeks, but not exceeding the maturity of the check, were classified as medium-early selections. Short-statured selections were those with plant heights shorter than 120 cm and medium- statured selections were those with heights between 120 cm but not exceeding 150 cm (height of the check Peta). Chlorophyll mutations, plants with sterile florets, and plants that were photoperiod sensitive occurred rarely. A total of 979 plant selections were harvested from the dry- season planting. The M4 plant-progeny selections were sown in an ordinary wet bed on July 8-9, transplanted on August 1 and cultured in the same manner as the M3 material described above.

2. Results

The number of selected plants and types of selections from the M2 and M3 generations of progenies of rice seeds irradiated with fast neutrons are indicated in Table III. Three hundred and eight plant selections from the M3 generation of BE-3 and BPI-121, and 85 from the M3 generation of Bengawan, Peta and Intan were selected. These are all early to medium-early and short to medium-short statured plants. More beneficial mutants were obtained with the lower doses of neutrons on BE-3, BPI-121 and Bengawan, and with the higher doses on Peta. Plant-progeny rows of these selections, except those from the photoperiod- sensitive varieties (BE-3 and BPI-121) are being grown this 1968 dry season for further evaluation of their agronomic characters. 92 VIADO et al.

TABLE III. NUMBER OF SELECTED PLANTS AND TYPES OF SELECTIONS FROM PROGENIES OF RICE VARIETIES IRRADIATED WITH FAST NEUTRONS 1967 wet-season harvest

Types of selections No. of plants Generation Variety and treatment Short Medium Medium- selected Early statured statured early

M, BE-3

6.6 X 10" n/cm2 55 35 20 - 55

•1.32 X 10u 45 45 - 1 44 . .1.78 X 1011 130 - 130 - 130 1.98 X10n 8 - 8 - 8

2.64 X10u 5 - 5 - 5

Total 243 80 163 1 242

BPI-121

6.6 X 1010 n/cm2 25 3 22 - 25

1.32 X 1011 24 3 21 - 24 1.78 X1011 5 5 5

1.98 X1011 5 - 5 - 5 2.64X10" 6 3 3 - 6

Total 65 9 56 - 65

M3 Bengawan

6.6 X 10" n/cm2. 10 3 7 7 3

1.32X10" 7 - 7 7

1.78X10" 8 - 8 4 4

1.98X10" - - - - -

2.64 X 10" 8 - 8 8- -

Total 33 3 30 26 7

•.. peta

6.6 X 10" n/cm2 2 - 2 2 1.32 X 10" 9 - 9 8 1

1.78 X 10" - - - - - 1.98X10" 8 - 8 3 5

• 2.64X10". 17 - 17 10 7

Total 36 - ' 36 . 21 15

Intan

1.32 X 1010 n/cm2 16 9 7 13 3

Total ~16

Grand total 393 101 292 61 332 VIADO et al. 93

TABLE IV. NUMBER OF SELECTED PLANTS AND TYPES OF SELECTIONS FROM M4 PROGENIES OF VARIETY PETA IRRADIATED WITH GAMMA RAYS AND MIXED RAYS OR TREATED WITH EMS 1967 wet-season harvest

Types of selections No. of plants Treatment Short Medium selected Early Medium-early statured statured

Gamma rays(kR)

1.0 - - - - -

fo.o 12 - 12 12 -

20.0 48 - 48 48 -

35.0 112 29 83 112 -

45.0 115 - 155 155 -

Total 327 . 29 298 327 -

Reactor mixed radiations (rads)

100 65 - 65 65 -

500 4 - 4 - 4

1000 82 - 82 82 - 2 000 19 16 3 16 3

3 500 60 - 60 60 -

5 000 21 - 21 21 -

10 000 4 - 4 - 4

Total 255 16 239 244 11

EMS (%)

0.0078 61 - 61 55 6

0.0625 28 - 28 28 -

0.125 67 - 67 67 -

0.25 35 - - 35 - ' 35 1.5 117 15 102 55 62 1.0 1540 6 1534 1497 43

Total 1848 21 1827 1702 146

Grand total 2430 66 2364 2273 157

In the M4 generation of Peta (Table IV) a larger number of short to medium-short and early to medium-early plants were selected from the higher doses of gamma rays (35 to 45 kg) and EMS (0.5 to 1.0%). There is no definite trend in the number of beneficial mutants as affected by different doses of mixed rays. A total of 2430 plant selections were harvested. These materials are now being grown in plant-progeny rows this dry season for further evaluation. Seventy-three uniform mutant lines were selected from the M3 generation of Peta and Intan after neutron irradiation and from the M4 generation of Peta after treatment with gamma rays, mixed radiation and EMS. These lines are all earlier and mostly shorter than the Peta variety (time to heading for the lines was 91-116 days, for Peta 120 days; plant height of the lines was 106-170 cm, for Peta 158 cm). Further tests are being made with the check varieties. CD

TABLE V. DAYS TO MATURITY, AVERAGE HEIGHT AND AVERAGE NUMBER OF TILLERS OF EARLY MATURING AND NON-SEASONAL LINES SELECTED

No. of lines selected a No. of lines Total of Average height Average No. of Variety Treatments Range of maturity Remarks planted lines selected (cm) tillers per hill fot further study for preliminary trial

BPI-121 1 kR y-rays 62 50 6 62 120-125 119.6 8.2 Control variety BPI-121 flowered earlier than the treated ones. 2.5 kR y-rays 78 50 6 78 120-125 138.2 10.0 This is due to late planting which Control. 118 139.0 7.6 indicates sensitivity to photoperiod.

Sum 140 100 12 140 - -

5 kR y-rays 197 50 8 159 125-130 104.4 12.6

7.5 kR yvrays 17 50 6 77 130-136 . 130.4 11.8 No uniform lines from BE-3 BE-3 10.0 kR y-tays 83 60 - 70 136-140 136.0 9.0 treatment with 10 kR were obtained for preliminary yield trial. 35.0 kR y-rays 140 50 6 140 125-130 143.0 11.8

1000 rads mixed rays 101 50 8 101 125-130 118.0 6.8

Control - 135 135 10.2

Sum - 598 260 28 547

Grand total 738 360 40 687

3 Planted for further study and preliminary yield trial during this "palagad" season. VIADO et al. 95

III. EXPERIMENTS AT THE MALIGAYA RICE RESEARCH AND TRAINING CENTER, MUNOZ, NUEVA ECIJA

The research program consisted of two parts: (a) Selection and testing of possible mutations obtained by treatment of the varieties BE-3, BPI-121, Raminad str.3, Elon-elon and Wagwag with gamma rays from 60Co, mixed radiations from the nuclear reactor, and ethyl methyl sulphonate (EMS). (b) Field tests of japonica and indica mutants from Taiwan and India.

RESULTS Experiment 1

Early maturing selections were made from gamma-ray and mixed- radiation treated BPI-121 and BE-3. The early maturing lines from BPI-121 and BE-3 were sown in the seed-bed on August 2, and transplanted on August 31, 1967. Table V shows the number of lines selected from plantings of early maturing lines from BPI-121 and BE-3 obtained after treatment with gamma rays and mixed radiations. From a total of 140 lines of BPI-121 from gamma-ray treatments with 1 and 2.5 kR, 100 lines were selected for further study. From BE-3, 260 lines were selected out of 547 lines obtained from 5.0, 7.5, 10 and 35.0 kR of gamma rays and from 1000 rads of mixed radiations. A total of 360 lines from these varieties were selected and planted during the 1968 dry season as M5 lines for further study of their photoperiodic sensitivity. From the two treatments of BPI-121 and the five treatments of BE-3, 12 and 28 early maturing lines respectively (i.e. a total of 40 lines) were selected and planted for preliminary yield trials as M5 during the 1968 dry season. The range of maturity, average height and average number of tillers per plant of the 140 lines of BPI-121 and 547 lines of BE-3 are also indicated in Table V. The 140 BPI-121 lines matured in 120 to 125 days. It may be noted that the selected early maturing lines of BPI-121 seemed to mature later than the control plants. This may be explained by the fact that the original BPI-121 used as control was a photosensitive variety so that when planted late in the season its length of maturity was considerably shortened. Hence, it was shorter in maturity than the non-seasonal selected lines. It is also interesting to point out that 62 lines out of 140 of BPI-121 were very much shorter than the control plants. The tillering capacity of the 140 lines was higher per plant than the control. The BE-3-lines selected from 5 and 35 kR and 1000 rads were earlier by 5 to 10 days than the control plants. Those that were obtained from the 7.5 and 1-0 kR treatments matured practically at the same time as the control. This is also due to the photosensitivity of the parent or control variety BE-3. In height, the 159 lines from the 5-kR treatment were very much shorter than the control plants. The lines obtained from the 5-kR treatment had the highest number of tillers among the selected plants while the lowest number of tillers per hill was obtained from the 1000-rad treatment. TABLE VI. LINES PLANTED, SELECTION MADE AND RANGE OF MATURITY OF LINES IN EACH VARIETY '

BPI-121 : 8E-3 .' Elon-elon- Raminad Str. 3 Wagwag

No. of No. ol . No. of No. of No. of .. Treatment Range Range Range Range Range Panicles Lines Panicles Lines Panicles Lines panicles Lines Panicles Lines of maturity of maturity • of maturity of maturity of maturity planted selected planted selected planted "selected planted selected planted selected

164-193 2.0 35 59 162-167 30 36 176-180 24 44 180-188 24 35 1.0 45 45 145-148 35 58 ' 162-163 24 60 _ 175-180 24 31 173-184 - - - 198-202 • 0.5 40 69 156-160 '35 62 162-163 24 32 179-180 24 59 , - - 18 37 182-198' 0.25 40 • '42 145-160 - ''40 59 162-163 24 28 176-183 24 - 37 181-186 30 28 180-183 24 32 197-204 EMS 0.125 • 60 56 157-160 '40 69 166-169 24 27 • 166-180 24 26 148-160 . 56 166-171 24 30 169-180 24 26 180-184 24 36 182-197 m 0.0156 35 47 . 35 197-202 ' 0.0312 40 65 130-160 35 64 176-179 24 50 169-180 24 ' 45 173-184 24 35 182-194 0.0625 - - - ' 35 35 176-180 24 26 . 175-180 - - - 24 34 180-184 0.0078 25 ' ' 27 147-160 45 37 142-166 . 33 173-179 24 . 68 " - 180-186 180-185 ' 6 180-188 6 225-229 Control 6 - 163-166 6 " 6 " '

1.0 25 34 149-156 25 42 168-173 40 35 - 178-179 35 . 43 183-186 30 • 51 ., 196-198 198-203 2.5 25 36 156-159 30 56 165-168 45 30 ' 170-175 15 54 184-186 45 57 196-202 5.0 25" .26 153-156 25 30 169-171 ' 30 36 163-179 30 61 179-186 35 40 35 ' 65 196-204 Gamma rays ,7.0. 25 .26 . 153-156 25 33 153-163 30 ' 45 - '172-179 .30 30 183-186 30 179-184 30 33 196-199 (kR)' 10.0 25 ' 42 " 159-163 35 . 45 169-174 30 28 169-179 36 196-202 20.0 30 37 ' 156-159 30 50 • 159-170 ' 30 ' 37 178-179 • 35 40 181-186 35 44 179-180 35 39 179-186 35 27 196-208 35.0 - 30 • . 47 " 163-170 15 31 173-179 30 48 163-168 186-189 6 191-192 ' 6 189-190 - 6 222-226 Control 6 " . 6 " " " "

100 30 39 155-159 30 36 162-165 40 31 169-177 30 60 182-185 35 37 189-191 157-159 30 39 '• 167-172 35 ' '171-178 30 36 184-187 40 29 186-190 ' 200 " 35 45 19 500 "35 "" '30 • 153-159 30 44 165-172 . 30 28 169-172 35 26 181-186 40 32 189-192 1000 40 '38 158-162 35 49 169-172 30 ^ 38 171-176 .35 41 183-188 35 41. 184-188 189-191- ' 2 000 30 >1 155-162 30 38 171-174 30 35 175-178 35 44 186-188 35 35 30 .. 64 186-189 Mixed radiations 3 500 30 32 ' 155-160 30 41 164-169 •30 39 169-178 35 32 178-184 182-188 35 43 189-191 (tads) 5000 30 52 158-162 30 37 165-177 37 63 .165-178 35 32 189-192 7 500 35 50 156-159 30 44 168-174 30 31 170-178 40 61 180-185 30 57 171-176 35 ' 179-183 40 67 182-187 . 10 000 15 46 156-159 35 ' 34 162-172 • 30 36 - 76 . 189-192 15 000 30 35 ' . 156-159 40 35 165-172 30 ^ -31 173-178 35 36 183-187 . 40 ' 43 6 172-177 173-180 6 189-194 6 226-228 Control 6'- 157-159 j " 6 , - - " VIADO et al. .97

Experiment 2. Selections from M3 after EMS and radiation treatments

The M3 seeds were sown from June 19.to 23, 1967. Transplanting began on July 18, 1967. The distance between the plants and the rows was 20 cm. The crop was fertilized with 80-50-50 kg/ha and top dressed with 20 kg N/ha. The number of selected panicles and the range of maturity of the lines from each dose of EMS, gamma-rays and mixed radiations are shown in Table VI. Further laboratory studies of the grains of each panicle will be made on these field selections. Very early maturing lines were obtained from BPI-121, BE-3 and Wagwag, and earlier maturing lines were also observed from the two other varieties. The differences in the length of maturity between the selected lines and the control for BPI-121,. BE-3 and Wagwag was about one month with the EMS treatment. Marked differences in length of maturity between treated lines and the control were not observed among the varieties treated with gamma rays and mixed, radiation, although the treated lines in most of the doses of each treatment matured earlier than the control. Observations were made on the number-of tillers, the plant height, length of panicles, weight of grains and sterility for all the selected M3 plants. These agronomic characters will be followed up in the next generation.

Experiment 3. Field tests of japonica and indica mutants from Taiwan and India 1

The plots in the yield trial of the indica group were replicated four times and the experimental design was the randomized block system. The japonica mutants were replicated twice. In both tests BPI-76 was used as the check variety. The results obtained in the yield trial and in the observational trial of indica and japonica mutants are presented in Tables VII and VIII respectively. The data represent the average of the replicates. To identify the strains and compare their performance see Rice Breeding with Induced Mutations, Technical Reports Series No.86, IAEA, Vienna (1968).

(a) -Yield trial of the indica group (Table VII)'

The, mean number of tillers of the mutants ranged from 8.7 to 14.1- per hill. Three of the varieties produced 14.0 to 14.1, while the majority produced 12 tillers per hill. In length of maturity, IR-154-77-2 was"the earliest with 82 days, followed by NH-X-^-131 mut. with 105 days from sowing the seeds. The majority of the varieties matured in-109 days. BPI-76 (NS), IR-8-288-3 and CP 231 X SLO 17 were the latest, all maturing in 124 days. The mean -height of the plants showed that a number of the indica group had short straw - the majority of the varieties did not grow higher than 1 metre, whereas the rest were taller (139.4 to 151.2 cm high). None of the varieties, in spite of tallness in some, lodged at maturity. CO OO TABLE VII. YIELD TRIAL OF indica MUTANTS DURING THE WET SEASON 1967-68

Grain Maturity Mean plant height Spikelet fertility Plot yield Variety Mean No. of tillers Lodging characteristics wt/100 grains Diseases Pests (days) (cm)

~~1. Taichung (N) 1 14.0 109 85.2 Non-lodging 47.37 2.19 280.3 BLB~~

~~2. CP 231 dwarf(Muc.) XRex 8.7 111 67.3 46.53 1.91 87.3 BLB. tungro Rats~~

~~3. CP 231 dwarf (Mul.) XTP 11.4 111 94.8 57.86 1.79 326.5 BS, BLB~~

~~4. CP 231 X SLO 11 10.8 124 80.1 53.29 1.98 137.8 BS. BLB~~

~~5. 1R-8-288-3 12.9 . 124 87.4 40.47 2.70 255.3 BS, BLB~~

~~6. 1R-154-77-2 12.2 102 82.7 64.05 2.08 241.8 BS. BLB~~

~~7. NM-N,-4 (Mut. of 9) 11.8 109 143.0 77.57 3.27 285.5 BS, BLB~~

~~8. NM-Xi-131 (Mut.of9) 9.8 105 139.4 63.14 3.17 222.5 BS, BLB~~

~~9. Nahng Mon S-4 12.0 109 151.2 87.02 3.40 275.5 BS, BLB~~

~~10. NP 130 Mut. S-l 14.1 109 92.9 60. 70 3.75 115.0 BLB Rats, stem borer~~

~~11. NP 130 Mut. S-3 14.1 109 93.1 53.77 1.81 118.5 BS, BLB, SB Stem borer tungro~~

~~12. Yuang-Hsing 1 a 13.3 109 84.0 61.50 2.20 290.3 BLB Stem borer~~

~~13. I-Kungbau 4-2 (Mut.) 13.0 109 76.6 37.92 2.09 166.8 BS, BLB Stem borer~~

~~14. Shung-Chiang 30-21 (Mut.) 12.9 109 82.6 62.36 2.22 211.8 BS. BLB~~

~~15. Keh-Tze 20-74 (Mut.) 12.9 109 84.3 55.85 2.18 268.3 BS, BLB~~

~~16. BPI-76 (NS) 12.0 124 108.2 70.48 2.00 241.3 BS, ELB~~

Date sown: July 13, 1967 Typhoons: Diseases: Date transplanted: August 9, 1967 Trining: Oct. 16, 1967 B: Blast Area: 720 m2 (yield trial and observational trial) Strong . BLB: Bacterial leaf blight * , Wei mine: Nov. 3, 1967 BS: Bacterial stripe Distance of planting: 30 cm x 20 cm. ° bn/h« VerV SU011g SB: Seedling blight Top dressing: 20-0-0 kg/ha

~~a Selection from [Taichung(N) 1 x Shung-chiang 30-21 (Mut.)] TABLE VIII. PERFORMANCE OF japonica MUTANTS IN THE OBSERVATIONAL TRIAL, REGULAR WET SEASON 1967-68~~

~~Grain Maturity Mean plant height Spikelet fertility Plot yield Variety Mean No. of tillers Lodging characteristics wt/100 grains (g) (days) (cm) (» . Diseases Pests (S>~~

~~1. Tainan 3 11.2 115 105.4 Non-lodging 84.3 2.5 475.5 BS, BLB~~

~~2. FF 36 12.7 109 83.9 56.3 2.2 313.5 BS Stem borer~~

~~3. Irrad. Taichung 65(Mut.) - 21.9 109 94.1 • 92.9 2.7 368.5 BS. BLB, tungro~~

~~4. Fujiminori 8.6 101 73.5 85.6 2.6 197.5~~

~~5. Fvikie 70 (Mut, of 4) 1.1 101 69.0 91.4 2.5 154.5 BS, BLB. SB~~

~~6. Fukie 71 (Mut. of 4) 7.2 101 41.9 48.8 2.3 91.0 BS, BLB, tungro~~

~~7. Koshihikare 10.4 101 61.9 83.3 2.4 130.5 BS, BLB~~

~~8. Norin 22 8.0 101 56.2 „ 67.2 2.5 74.0 BS, BLB~~

~~9. R-90 (Mut. of 7) 10.6 101 61.7 83.2 2.4 105.5 BS~~

~~10. San-in 73 (Mut. of 8) 8.8 101 66.4 81.4 2.8 127.5 BS, BLB, tungro~~

~~11- 'Allorio 11 Temoin 6.4 102 104.2 Lodging r 71.4 ' 2.9 113.0 BS. BLB 12. Allorio Lambda (Mut.ofll) 9.4 101 76.1 Non-lodging 78.6 2.9 141.0 BS, BLB, SB~~

~~13. BPI-76 (NS) 11.5 124 53.5 68.5 1.9 322.0 BS~~

Date sown: July 13, 1967 Typhoons: Date transplanted: August 9, 1967 Tciiung-.. Oct. 16, 1967 Blast Area: 720 m2 (yield trial and observational trial) Strong Bacterial leaf blight Distance of planting: 30 cm X 20 cm Welming: Nov. 3, 1967 Bacterial stripe Fertilizer applied: _ , rn . „ Very strong Seedling blight rr Basal: 80-50-50 kg/ha Top dressing: 20-0-0 kg/ha 100 VIA DO et al.

The highest figure for spikelet fertility (87.02%), was obtained from variety Nahng Mon S-4. This was followed by variety NM-Ni-4 mut. with spikelet fertility of 77.57%. BPI-76 (NS) Ck gave only 70.48% spikelet fertility, and four of the test varieties produced less than 50%. . The seed weight per 100 grains showed variability. As shown in Table VII, the weights for three varieties were 3.40, 3.27 and 3.17 g per 100 seeds. On the other .hand, five varieties .had seeds weighing from 1.75 to 1.98 g per 100 grains, whereas the majority of the varieties gave grains that weighed 2.00 g per 100 grains. It is interesting to note that Nahng Mon S-4, the variety that had the highest spikelet fertility, has also the highest seed weight per 100 grains. The yield per plot of 1.5 m2 shown in Table VII may have been affected by the two typhoons, Trining and Welming, which occurred on October 16, 1967 and November 3, 1967, respectively. The data on yield per plot of the 16 entries in the test of indica mutants show that eight were comparable in productivity with the check variety, BPI-76 (NS). In fact, seven of the eight varieties produced higher yields than the check variety, particularly CP 231 dwarf X Rex with a yield of 326.5 per plot compared with 241.3 g per plot of BPI-76 (NS). The diseases that were common in most of the varieties were bacterial leaf blight and bacterial streak. Apart from these diseases, tungro was observed in two varieties, NP 130-mut S-3 and CP 231 dwarf X Rex. Rats and stem-borers were also found attacking the plants. NP 130-mut S-l and CP 231 dwarf X Rex were more severely attacked by rats than the other varieties.

~~(b) Observational trial of japonica mutants~~

The results of the observational trial of japonica mutants during the wet season are presented in Table VIII. More japonica mutants had less tillers per hill than the indica mutants. The lowest number of tillers among the japonica mutants was 7.4 and the highest was 21 tillers per hill. ' The length of maturity for 9 of the 12 mutants was 101 and 10 2 days from sowing the seeds in the seed-bed. The mean plant height of the varieties ranged from 41.9 to 105.4 cm. Only Allorie 11 Temoin, which was 104.2 cm, lodged at maturity. The spikelet fertility was generally higher among the japonica mutants than among the indica group. Eight of the 12 mutants had higher spikelet fertility than the check variety - it ranged from 71.43 to 92.94% compared with the check BPI-76 which had only 68.51% fertility.. There were no applicable differences in the grain weight among the mutants. The yield of the different mutants varied considerably. The lowest yielder, Norin 22, gave only an average yie^ld of 74.0 g per plot while the highest yielder, Tainan 3, produced 475.5 g. Of the twelve mutants only two produced higher yields than the check variety. Similar to the indica mutants in the yield trial, the japonica group showed infection by bacterial leaf blight and bacterial streak. Some of the mutants were also infected by tungro. VIADO et al. 101

~~SUMMARY~~

One hundred and forty non-seasonal lines were selected from BPI-121, treated with 1 and 2.5 kR. The range of maturity of the selected lines was 120 - 125 days from sowing to harvesting. In BE-3, 547 lines were selected for non-seasonal characteristics with a range of maturity from 125 to 140 days. Of these selections twelve from BPI-121 and twenty-eight from BE-3, which were of practical uniformity, were ele- vated to the preliminary yield test during the 1968 dry season. From the different varieties .treated with different mutagens a large number of panicles has been selected for further studies. Some of these selections were early in maturity compared with the control plants. Observations on some of the important characters, particularly the number of tillers, height, length of panicle, seed weight and percentage of sterility were also made. Data on these characters show that there seems to be no definite trend on the effects of the three treatments in relation to the dosage used. Field trials with mutants of indica and japonica rice from Taiwan and India gave the following results:

(a) Two of the indica mutants had a higher percentage of spikelet fertility than the check BPI-76 (NS). • (b) Eight of the indica mutants were comparable in yield with the check variety BPI-76 (NS). In fact, seven produced higher yields than the check variety. • (c) The range of maturity of the indica mutants was from 102 to 124 and all. of them were non-lodging. All the mutants in the test as well as the check variety were affected by diseases, particularly bacterial leaf blight and bacterial streak. Tungro disease was also observed in two mutants. (d) In the observational trial, the. range, of maturity of the japonica mutants was 101 to 109 days; the height of the plants varied from 41.9 to 105.4 cm; all mutants were non-lodging, except Allorio 11 Temoin which lodged at maturity. Two of the mutants, FF 35 and irradiated Taichung 65 Mut., produced higher yields than the check variety.

~~IV. EXPERIMENTS AT THE COLLEGE OF ARTS AND SCIENCES, UNIVERSITY OF THE PHILIPPINES, DILIMAN, QUEZON CITY~~

~~Selection of eating quality in Mg IR-8 rice (Experiment No. 067213)~~

The IR-8 rice variety, popularly called "miracle rice", is about the best rice plant in the world today due to its high yield and dwarf growth habit. These two qualities overshadow its highly specific cultural requirements, like nitrogen-rich soils, plenty of irrigation water and effective weed and pest control. Its main drawback, however, is its poor eating quality which makes this variety a second class commercial rice on the market. Hence, the reason for this project to improve the eating quality of the IR-8 variety through induced mutation and selection. 102 VIADO et al.

~~Method~~

Gamma irradiation of the seeds and growth of the Mi generation have been described in previous reports. This paper deals with the selection test for eating quality in the M2 generation. The M2 crop was planted in a three-seeds per Mi plant plan. The plants were harvested line by line and the yield per line was threshed by hand and stored for three months in labelled paper bags. After the storage period, the grains were divided into two lots of equal amounts. One lot was used for the cooking test and the other for M3 planting. The grains for the cooking test were cleaned by pounding them by hand in a wooden mortar and pestle as the rice mills refused to clean small amounts of seeds. When most of the grains of the M2 lines had been cleaned, the cooking test began. The kernels were cooked in small pots over earthen electrical burners. As far as possible, care was taken to make the cooking conditions the same with respect to cooking temperature, duration of cooking, amount of water for boiling the rice and period of cooling the cooked rice for the test. The eating quality test (EQ) was done when the cooked rice got cold, because the cooled cooked rice of IR-8 differs from the ordinary commercial varieties. There is hardly any difference in eating quality in newly cooked rice. Eating quality was scored as soft (s), medium hard (s+) and hard (+). These qualities were based on the quality of the standard, the Peta variety, and that of the normal, unirradiated IR-8 rice.

~~TABLE IX. EATING QUALITY AND COLOUR OF COOKED RICE IN M2 IR-8 VARIETY~~

~~Gamma-ray dose Colour of ^ , . ,, No. of M2 lines Eating quality Culture No. and variety on Mi ° cooked rice , tested (kR)~~

~~142 Peta 0 102 102 0 0 102 0~~

~~143-1 IR-8 0 180 0 0 100 0 180~~

~~143-2 IR-8 10 203 2 0 201 4 199~~

~~143-3 IR-8 20 264 1 3 260 1 263~~

~~143-4 IR-8 30 236 1 2 233 6 230~~

~~143-5 IR-8 40 285 1 5 279 2 283~~

~~143-6 IR-8 50 207 2 1 207 3 204~~

~~Legend: s - soft ccld cooked rice s+ - medium hard cooked rice (cold) + - hard cold cooked rice~~

~~- white cooked rice - light or pale brown cooked rice VIADO et al. 103~~

The colour of the IR-8 cooked rice was also tested. The rice was either pale to light brown or white. This test is only applicable to hand- milled rice. With machine-milled IR-8, the colour of the cooked rice is white like the commercial varieties.

~~Observations and results~~

As shown in Table IX, out of 1192 M2 lines tested so far, only about 0.59% had soft cold-cooked rice compared with the Peta standard. Ap- proximately 0.89% had medium-hard cold-cooked rice, and the rest had an eating quality like the normal IR-8 variety. The 7 lines with soft cooked rice and 11 lines with medium-soft rice were obtained at random without any particular Mi radiation dose association or correlation.

~~This may be due to the fact that not all the M2 lines have been accounted for.~~

~~Future plans~~

The few selected lines with soft cold-cooked rice and white colour of cooked rice will be grown in the M3 generation during the regular rice growing season in June-December, 1968. Planting of these "good" lines is desirable in relation to problems of adaptability and highest yield under normal rice growing conditions.

~~INDUCTION OF MUTATIONS IN THAI RICE VARIETIES AND SUBSEQUENT SELECTION AND TESTING . OF BENEFICIAL MUTANT LINES~~

~~S. DASANANDA, P.'KHAMBANONDA Ministry of Agriculture, Bangkok, Thailand~~

~~Abstract~~

INDUCTION OF MUTATIONS IN THAI RICE VARIETIES AND SUBSEQUENT SELECTION AND TESTING OF BENEFICIAL MUTANT LINES. Ionizing radiations were first used in the Thaiiand Rice Breeding Program in 1955 when seeds of two recommended varieties were sent to the United States of America for treatment. As a result, five promising mutant lines are at present in regional yield tests where" they are being considered for recommendation to rice growers. During the period 1960-1961 an unsuccessful attempt was made to induce resistance to blast.in three susceptible varieties by exposing seeds to a local source of ionizing radiation. In 1964, after an elapse of about 4 years, anqther.-attempt was made to utilize ionizing radiations in the breeding program by treating seeds of two recommended varieties. In 1965, a co-ordinated rice mutation breeding program was initiated under the auspices of the Joint FAO/IAEA Division of Atomic Energy in Food- and .Agriculture which resulted in treating seeds of twelve different rice varieties with both ethyl methane • sulphonate arid gamma'rays from a 60Co gamma cell. The results so far indicate that mutagenic agents have been successful in producing genetic variability.

Differences in heading date, mature plant height and plant type are frequently observed in the M? and M3 generations. Several lines obtained from two of the-irradiated varieties have exhibited a higher degree of resistance to blast than the parental material,. From 15-kR treatments,of non-glutinous varieties, mutants with glutinous endosperm have been obtained. Not all varieties gave the same response to treatment.

~~INTRODUCTION ' ' : ;~~

Thai rice is characterized by its long slender and clear grain which, when cooked, becomes fluffy and remains soft for a relatively long time. Hence, Thai rice has become noted throughout the world for its superior quality. Although a number of varieties with excellent grain quality exist there is a need to irhprove the plant by developing types with shorter' straw, better resistance to disease and better response to fertilizers. Attempts to effect'variety improvement have been made through irradiation in addition to the conventional'breeding program. • In 1955, rice seeds of two recommended Thai varieties were sent to the United States of America to be treated with X-rays and thermal neutrons in an attempt to obtain superior varieties by mutation breeding. As a result, five promising mutant lines of the two irradiated varieties are at present included in advanced yield trials before being considered for release. The mutant lines have exhibited better straw strength and higher | yield than the two parent varieties in preliminary tests! In addition, experiments were initiated in 1960 to induce blast resistance in three susceptible varieties by irradiation of seed with gamma rays at Kasetsart Univer- sity, Bangkok, Thailand. Unfortunately, induction of blast resistance was not achieved at that time because of some technical problems. Since 1964,

105 106 DASANANDA and KHAMBANONDA after an elapse of about 4 years, rice seeds of several varieties were again treated with gamma rays at the Thai Research Reactor No. 1. In 1965, a co-ordinated rice mutation program was initiated in Thai- land under the auspices of the Joint F AO/IAEA Division of Atomic Energy in Food and Agriculture. Gamma rays from a 60Co source contained in a cell were used to treat rice seeds at the Thai Atomic Energy for Peace in 1966. Induced mutations by the use of chemical mutagen, ethyl methane sulphonate, to treat rice seeds were started in 1966. Thus, the present joint program, which was started in 1964, is still in progress.

~~MATERIALS AND METHODS~~

Experiment I. Started in October 1964. Irradiated varieties: Nahng-Mon S-4 and Meuy-Nawng 62 M. Moisture content of seeds: About 14%. Ionizing radiation: Gamma rays from 235U source of Thai Research Reactor No. 1. Dosage; 15 and 30 kR at 1 kR per minute.

Experiment II. Started in July 1965. Irradiated varieties: Niaw-San-Pah-Tawng, Khao-Dawk- Mali 105, Leuang-Pratew 28, Khao-Tah-Haeng 17 and Nahng-Phaya 132. Ionizing radiation: Gamma rays from 235U source of Thai Research Reactor No. 1. Dosage: 15, 20, 25 and 30 kR at 5 kR per minute.

Experiment III. Started in August 1966. Irradiated varieties: Khao-Tah-Haeng 17 and Leuang Awn 29. Ionizing radiation: Gamma rays from 235 U source of Thai Research Reactor No. 1. Dosage: 15, 30 and 45 kR at 5 kR per minute.

Experiment IV. Started in September 1966. Irradiated varieties: Bang-Khen 293, Khao-Dawk-Mali 105, and Khao-Pahk-Maw 17. Ionizing radiation: Gamma rays from 60 Co source of the gamma cell at the Thai Atomic Energy for Peace. Dosage: 8, 16, 32, 48 and 64 krads at about 15 krads per minute.

Experiment V. Started in October 1966. Treated varieties: Niaw-San-Pah-Tawng, Khao-Dawk- Mali 105 and Nahng-Phya 132. Chemical mutagen: EMS (ethyl methane sulphonate) Treatments: 0.1, 0.2, 0.3, 0. 4 and 0. 5% solution for 6 and 18 h. 107 DASANANDA and KHAMBANONDA

~~Pretreatments: Soaking .brown rice in water at room temperature 4 h for 6-h EMS treatment and 3. 5 h for 18-h EMS treatment.~~

Experiment VI. Started in January 1968. Treated variety: IR-8. Chemical mutagen: EMS Treatments: 1 and 2% solution at 22°C for 20 h. Pretreatment: Soaking in water for 24 h at 30°C before EMS treatment.

~~Mi generation~~

1. Seed (breeder seed) that had been exposed to physical (ionizing radiation) and chemical treatments was sown in pots or in seed-beds and transplanted one plant per hill in pots or field with close spacing of 10 cmX 25 cm apart'so that only a few tillers were, obtained. Untreated seed of the same seed source was grown as a control every tenth row. Survival rates at seedling and heading stages were recorded for both treated and untreated seed.

~~2. Panicles should be covered with pollination bags before spikelets start opening; however, bagging is a laborious job so the Mi was grown in an isolated field several metres from other varieties.~~

3. Panicles were bulked from each plant but plants were kept separately. Sterility percentage was recorded as the ratio of the number of sterile spikelets to the total number of spikelets in the panicle.

~~M2 generation~~

1. Seed from each Mj plant was grown in an upland seed-bed (short row) to test for blast resistance. Since some of the Mj plants may produce a considerable amount of seed, several short rows from each Mi plant may be sown, but all the seed was planted.

2. Only those seedlings that were blast resistant were saved and transplanted in the field at one seedling per hill with a spacing of 25 cm X 25 cm. The untreated variety was grown every tenth row. After the seedlings had been transplated for one month, all the plants that showed yellow-orange leaf virus symptoms were discarded.

~~3. At harvest time, the plants that had good plant type and other desirable characteristics were saved. Selected plants were kept separate.~~

Thus, in the M2 generation, we expect to save only those plants that have both virus and blast resistance and which exhibit good agronomic characteristics. A few seeds of each selected plant should be grown in tjie dry season to determine whether mutations have occurred for photoperiod insensitivity. 108 DASANANDA and KHAMBANONDA

~~M3 generation . • , .~~

1. The upland short row test for blast was repeated as in the M2 generation, but only 8 g of M3 seed from each selected M2 plant was used. Only those seedlings which were resistant to blast were transplanted at a spacing of 10 cmX 25 cm, with one plant per hill. Yellow-orange leaf disease symptoms were observed one month after transplanting in the field. One row of the untreated check was grown every tenth row.

2. Each row that was uniform was harvested in bulk. If plants were segregating in the rows, they were harvested separately and grown as pedigree plant rows in the next generation. Plant height and heading date were recorded in this generation. Promising mutant lines will be given a selection number in the order of heading date and height, respectively.

~~3. Especially promising lines which showed non-segregation were ' put in the uniform yield trial and grown at several stations to obtain information on their performance.~~

~~RESULTS~~

Experiment I. The M4 generation of both irradiated varieties was planted in the 1967 wet season. Selection for better blast and yellow-orange leaf virus resistance was practiced during the test period. For Muey-Nawng 62 M, screening for gall midge resistance was also made at the Sakolnakorn Experiment Station, located in the Northeast of Thailand where gall midge is usually a threat to rice production. Selection of desirable characteristics and good plant

~~type with intermediate height for M5 planting was made as follows: Variety No. of M4 selections planted No. of selections saved~~

~~Nahng-Mon S-4 543 rows 33 lines Muey-Nawng 62 M 315 rows 123 lines~~

The lines of both varieties will be planted in ' observation nurseries' to study the yielding ability and other performance in more detail in the next wet season. Some selected lines of Nahng-Mon S-4 mutants showed good plant type with slightly shorter straw and earlier maturity than the original variety. All the late maturing types were discarded from both irradiated populations.

Experiment II. The M3 generation of the five irradiated varieties were planted in five rice experiment stations in various regions of the country. Mutants with good plant type and other desirable characteristics from each irradiated variety were selected for either plant-to-row or observation nursery in M4, depending upon whether the selected M3 109 DASANANDA and KHAMBANONDA

~~rows appeared uniform in height and heading. Disposi- tion of the material was as follows:~~

~~Rice No. of M3 No. of Variety experiment plants selection station grown saved~~

Niaw- San- Pah- Tawng Sanpahtawng 737 rows 97 lines Khao-Dawk-Mali 105' Pimai 2 141 rows 2 141 lines Leuang-Pratew 28 Koksamrong 1 386 rows 690 lines Khao-Tah-Naeng 17 Supanburi 1 502 rows 286 lines Nahng-Phya 132 Kuangut 781 rows 53 lines

Two lines selected from the irradiated variety Niaw-San-Pah-Tawng headed approximately 2 weeks earlier than the original variety. Several other mutant lines of this variety exhibited wide variation in height and heading dates. Three lines of Khao-Dawk-Mali 105 from the 15 kR treatment may have mutated for endosperm characteristics resulting in a change from non-glutinous to glutinous rice. These glutinous mutant seeds will be increased and evaluated more thoroughly in the next season. Since most of the plants did not exhibit genetic variation all lines of this 'irradiated variety will be replanted in the 1968 wet season. Several Khao-Tah-Haeng 17 and Nahng-Phya 132 mutant lines showed better blast resistance than the original varieties. Nine Khao-Tah-Haeng 17 mutant lines also have glutinous endosperm.

~~Experiment III. The M2 generation of the two irradiated varieties was planted in the 1967 season. The number of plants collected~~

~~for M3 planting were as follows:~~

~~Variety No. of plants grown No. of plants selected~~

~~Khao-Tah-Haeng 17 62 rows 16 plants Leuang-Awn 29 6 5 rows 30 plants~~

~~Very few rows of either irradiated variety exhibited evidence of sterility and/or abnormal plants. Some genetic variation in height and heading dates was observed among and within the rows.~~

~~Experiment IV. The M2 generation of three irradiated varieties was grown in the 1967 season and the following number of selections • were made for M3 planting:~~

~~Variety No. of plants grown No. of plants selected~~

~~Bang-Khen 293 200 rows . 65 plants Khao-Dawk-Mali 105 141 rows 24 plants Khao-Pahk-Maw 17 169 rows 29 plants 1240 DASANANDA and KHAMBANONDA~~

A few rows exhibited evidence of sterility and/or abnormal plants, except in the irradiated variety Bang-Khen 293 which showed a high sterility and a heavy infection of sheath blight disease. Some genetic variations in height and heading dates occurred among and within the rows.

~~Experiment V. The M2 generation of the three varieties treated with EMS was planted in the 1967 season, and the following~~

~~were selected for M3 planting:~~

~~Variety No. of plants grown No. of plants selected~~

~~Niaw-San-Pah-Tawng 16 rows 3 plants Khao-Dawk-Mali 105. 26 rows 3 plants Nahng-Phya 13 2 24 rows~~

Very little genetic variation was observed in this material. Only three plants from each of the two irradiated varieties were saved for further studies in the M3 generation. The chemical mutagen treatment for rice should be conducted again with various treatment techniques and also on a wider scale.

~~Experiment VI. The M^ generation of the EMS treated variety was planted in the 1968 dry season. The following were collected for~~

~~M2 planting: No. of plants No. of transplanted No. of plants Variety grown seedlings collected~~

~~IR- 600 112 70~~

~~There were no differences in sterility percentage of the treated and untreated plants of IR-8. However, the survival rates at seedling and heading stages of the treated seed were markedly reduced.~~

~~DISCUSSION~~

K.I. SAKAI: You have described that in one experiment you failed to find mutants resistant to blast disease, while in the second experiment you succeeded. Can you tell me what is the reason for the failure in the first experiment and the success in the second? P. KHAMBANONDA: The success of the second experiment may be due to the better screening techniques and/or the bigger size of the Mj plant population than the one used in the first experiment. In the second experiment we used all the seeds from the M2 plant and sowed them in an upland short-row seed bed. Plants with good type and better blast resistance were selected in the M2 and M3 generations. M4 seeds of each selected plant were tested for blast resistance in the M4 generation. We have found several mutant lines with a higher degree of blast resistance than that of the original variety. RICE STEM BORERS IN MALAYA

~~A proposal to use mutation breeding for their control~~

~~F.C. VOHRA University of Malaya, Kuala Lumpur, Malaysia~~

~~Short communication~~

~~1. The problem of rice stem borers~~

Among the various problems in the rice crop the loss caused by the larvae of lepidopterous stem borers seems to be very serious. In some districts of the Kurian area, the rate of damage has been as much as 100% [ 1]. It is not unlikely that the stem borer threat will become worse with the introduction of double cropping in many areas. Research into stem borers and efforts for their control date back to 1930 when Pagdon [ 2] published his paper "A preliminary account of three rice stem borers". Several later papers described natural parasites of stem borers and greatly emphasized their use for biological control of the borers. Thus Trichogramma namum Zehnt was tried but with little success. Another futile attempt was made with an imported species of a Tachinid fly Para- theresia claripalpis de wulp after the war in 1951. Since then the control of stem borers has mainly been through the application of various insecti- cides, namely DDT, BHC, Endrin, dieldrin and gamma-BHC, and this has met with a certain amount of success. The chemical method of control, however, is not fully satisfactory because of:

(a) The high recurrent cost in application; (b) The danger of hazard to man by residues; (c) The toxic effects to fish and mammals in the paddy fields [ 3]; (d) The poisoning of the natural parasites of the borers; (e) The difficulties for proper application, exact concentrations etc. by untrained farmers; and (f) The probability for development of insecticide resistance in stem borers.

Apparently there is an urgent need for some control free from chemical hazards. Scientists in various countries have turned to breeding for varietal resistance, among other approaches, to save the crop from damage by stem borers. The development of a variety of rice resistant to stem bores will provide an effective control operative at all levels of insect population without additional cost and inconvenience to the farmer..

~~Ill 112 VOHRA~~

~~2. The research project - -~~

To achieve this objective a research project has recently been started as a co-operative venture at the School of Biological Sciences, University of Malaya, Kuala Lumpur. This project will involve: " •

(a) Detailed ecological studies of borers in different varieties of rice grown commonly in the various parts of the country; (b) A study of parasites of borers for possible biological control; and (c) Production of rice varieties resistant to stem borers...through induction of mutations and hybridization with locally used types. '

In Malaya only the following four lepidopterous stem borers are important [4, 5] : Sesamia inferens (Wlk); Chilo suppressalis (Wlk); Chilotraea polychrysa (Meyer); and Tryporyza incertulas (Wlk). Various factors pertaining to the morphology, physiology and chemical nature of the rice plant are possibly responsible for the stem borer resistance. Carefully planned experiments have to evaluate their contribution to resistance before successful breeding programs can be undertaken. A wild species of rice in Malaya, Oryza ridleyi Hook, is said to-be highly resistant [6]. Similarly, several cultivated varieties are claimed to offer considerable resistance, but none of them can be recommended for commercial planting because of unsatisfactory yield, grain size and appearance, and nutrient and cooking value. Therefore, in order to develop a valuable resistant variety one would have to improve all the a:bove characteristics. Muta- tion breeding with a high standard variety may be more promising than a long-term cross-breeding program and should, in the opinion of the author, also be attempted in other countries. ' '

~~3. Work in progress ••~~

Through the co-operation of the authorities of the Serdang Agriculture College, the use of their paddy fields for observation and experimentation has been secured. Preliminary work to assess the type of borer and infestation in the most common rice varieties namely Mahsuri, Malinja and Ria is already in progress. This is essential because most of the previous work on stem borers in Malaya has been carried out on older varieties which are not much favoured either by the government or by the people. All the four species mentioned earlier have been found attacking the new varieties at Serdang. At present, the data are too meagre to warrant any definite conclusions, but the following possibilities are indicated by the field observation.

(a) Some species of borers are more common in one locality than other- species. , (b) In a particuler locality there seems to be a quantitative host, variety preference. (c) There is no indication of a relationship between the borer infestation and the habit of the plants. (d) Some varieties are possibly more vulnerable during younger stages. (e) There is an indication of one species of borer being more abundant during early stages of the plant and another one during later stages. (f) There are many borers per tiller during the early stages of paddy plants, but only one or two at later ones.. VOHRA 113

These observations indicate some possibilities for resistance breeding which will soon be tested on new strains in the field and in the laboratory. For improving screening techniques on large plant populations, experiments are being carried out to improve methods of mass breeding of stem borer larvae in the laboratory. Besides this, the collection of parasites of stem borers is being attempted.

~~REFERENCES~~

[1] KAWASI, D., "Rice pests and their control", Rice Culture in Malaya, Symposium series No. 1, The centre for Southeast Asian Studies, Kyoto University (1965) 50. [2] PAGDON, H.T., A preliminary account of three rice stem borers, Sci. Ser. Dept. of Agric., Straits Settlements and Federated Malay States, Bulletin No.l (1930). [3] PATHAK, M.D., Ecology of common insect pests of rice, A. Rev. Ent, 13 (1968) 257. [4] KOK, L. T., VERGHESE, G., Yield losses due to lepidopterous stem borer infestations of Padi (Oryza sativa). Trap. Agric., Lond. (1965) 69. [5] YUNUS, ROTHSCHILD, C.H.L., "Insect pests of rice", The Major Insect Pests of the Rice Plant (Malaysia Symp.) IRRI (1967) 617. [6] VAN, T.K., GOH, K.G., The resistance of Oryza ridleyi Hook, to padi stem borer attack, Malay, agric. J. 42 (1959) 207.

~~DISCUSSION~~

K. I. SAKAI: With regard to your ecological studies on the rice stem borer, can you tell me if there is an ecological differentiation in forms, subspecies or other subdivision of the stem borer? F. C. VOHRA: To my knowledge there are no distinct subspecies of stem borers. The four species commonly occurring in Malaya do exhibit some ecological differences which are at present under detailed study. H. ISHIKURA: In connection with your statement that the dominant species of the four stem borers in Malaya differ from place to place, I would like to point out that the dominance of a given stem borer species seems to be related to the general environmental conditions, availability of alternative hosts (including crops other than rice) and the type of rice cultivation. Among these, the type of rice cultivation is expected to change remarkably in many rice-growing areas by the introduction of double cropping and fertilization at high level. I suspect that Chilo may become dominant over Tryporyza in areas where double cropping of rice may be adopted. This already happened in Taiwan some time ago. Observations have been made in various countries on the way in which several, species of stem borers bore into the rice plant tissue. These observations indicate that the method of attack depends on the rice species and on the developmental stage of the rice plant. In trying to breed rice varieties that are resistant to stem-borer attack, it would i>e necessary to take these differences into consideration. Some existing varieties, which are susceptible to the stem borer in terms of percentage of culms infested, do not give reduced yields.

~~RECOMMENDATIONS~~

~~ROLE OF INDUCED MUTATIONS IN RICE BREEDING~~

~~It was emphasized that induced mutations play a significant role in rice breeding.~~

~~The advantages of mutation breeding are:~~

1. Favourable specific changes in individual characters, without altering the rest of the genotype, can be obtained in an established variety. Such characters may be short culms, resistance to lodging, resistance against diseases, resistance against insect pests, improvement in grain quality (protein and amino acid content), and earliness, etc. 2. Polygenic changes for the improvement of quantitative characters, like yield, can be induced. 3. Additional variability'and synergistic effect may be obtained by subjecting the Fj seed to mutagenic treatment, particularly in inter-racialcrosses.

~~The disadvantages of mutation breeding are:~~

1. Direction and nature of induced variation are still largely at random. 2. It is not possible to expect desirable changes in more than a few characters at a time in the same individual unless the characters are controlled pleiotropically. 3. Most induced mutations are of negative selection value and hence a large population is needed for the detection of desirable mutations. 4. Induced mutations and hybridization methods are both important in rice breeding programs, although in specific cases one or the combination of the two may.be preferable, depending on the breeding goal and the genetic variation in the breeding material.

~~OBJECTIVES OF RICE MUTATION BREEDING PROGRAMS~~

~~I. Mutation breeding programs should aim at the development of superior varieties with the following characteristics:~~

1. High yielding ability: the high yielding ability of a rice variety depends primarily upon its nitrogen-responsiveness and lodging resistance. The following plant characteristics constitute basic requirements for such a variety: a. short stature b. strong straw c. erect, dark green and thick leaves of intermediate length and width d. early vegetative vigour e. slow leaf senescence f. high grain density and high grain weight.

~~115 116 RECOMMENDATIONS~~

2. Grain shape and quality: grain shape and cooking quality should be acceptable to the consumers of the different countries. An effort should also be made to improve the nutritional value of the rice by increasing the protein content and improving the amino acid composition.

3. Resistance to diseases and insects: varieties resistant to the following diseases and insect pests should be evolved: a. blast b. various virus diseases c. various bacterial diseases d. stem borers e. plant hoppers f. gall midge. It has been shown that genetic resistance to these plant adversaries can be developed by breeding.

4. Other characteristics: a. different maturity durations b. reduced sensitivity and insensitivity to photoperiod c. seed dormancy d. resistance to grain shattering e. drought resistance f. salinity resistance .

II. The best available varieties with wide adaptability should be used in mutation breeding programs. III. Exchange of breeding material and mutants between different breeders should be encouraged. IV. Mutation breeding should be used along with conventional breeding methods, depending upon the objective of the breeding program.

~~METHODOLOGY~~

~~Mutagen treatment~~

~~1. Ionizing radiations~~

~~Dormant seeds with a water content of about 10 - 14% should be used.~~

~~a. X- and gamma radiation The optimum dose range will normally be as follows: For indie a varieties: 15-30 krads For japonica varieties: 15-25 krads~~

b. Neutron radiation Both fast and thermal neutrons can be used. The dose range for rice is approximately 1/10 of the gamma dose in rads. Neutron radiation services are available at the IAEA Laboratory at Seibersdorf. Preliminary results from neutron irradiation in rice look promising and the group felt that a co-ordinated study RECOMMENDATIONS 117

fc» on comparative effects of fast neutrons and gamma radiation should be carried out. The radiation treatments should be done at the Seibersdorf Laboratory and the participants should send samples of one variety which would be given 3 doses of fast neutrons and gamma radiation.

~~2. Chemical mutagen treatments~~

Ethyl methane sulphonate has proved to be a highly efficient mutagen in rice. The use of diethyl sulphate should also be considered in practical mutation breeding programs. The seeds should preferably be presoaked in distilled water before mutagen treatments. The treatment time can thus be reduced and less injuries or physiological damage to the Mj plants are produced. No buffering of mutagen solution is necessary, but post-washing in running water is recommended.

~~3.. Combined radiation and chemical treatments~~

• Although interesting results have come out of combined treatments, it was felt that more investigations are needed before combination treatments can be recommended in practical rice breeding programs.

~~4. Handling of the treated material and successive generations~~

A minimum of 500 seeds should be used in each treatment. They should be seeded in a nursery or seeding box and transplanted in the field. The size of the Mi population depends on the breeding objective but, in general, a minimum of 250 - 300 plants should be used. The first three panicles should preferably be bagged to prevent out- crossing, or each variety should be isolated from other varieties.

From each Mx panicle, 15 -25 seeds should be planted out in rows for analyses and screening of qualitative, visible mutants in M2. If more variability is required, the number of Mj plants should be increased, and the Mj panicle selection and the number of plants to be grown in the

~~M2 on Mj plant basis could be suitably reduced. When quantitative polygene mutations are desired, special screening methods should be applied.~~

~~IMPROVEMENT OF FORMERLY POPULAR LOCAL VARIETIES (RESTORATION)~~

The group recognized that most emphasis should be placed on irradiation of new varieties which are deficient in a few characteristics. Characteris- tics of the variety under consideration should be carefully studied before undertaking a mutation program. If the variety is deficient in many characters it should not be used. The following steps were suggested:

1. Carefully select the mother variety on the basis of well-defined objectives. 2. Recognize that characters other than the ones under consideration will be changed. This means that a constellation of different 118 RECOMMENDATIONS i characters will be expressed. Therefore, it will be necessary to grow a large population in order to select those containing the most desired traits. 3. Consider utilizing only those characters relatively unaffected by environment, e. g. maturity, height, disease- and insect resistance, and grain quality.

It is recognized that certain varieties have become widely accepted by farmers and that the primary reason why they are no longer grown is the defects in one or a few characters. It is sometimes difficult to change the farmers' preferences despite a new variety being high yielding. The quality, plant colour, height, and smell of cooked grain, for example, tend to attract certain groups of farmers. Also, there are special conditions, such as deep water areas, where only floating varieties can be grown. Under certain circumstances the induction of a character, such as resistance to virus or production of short straw, may save an other- . wise popular, well-adapted variety.

~~QUALITY IMPROVEMENT~~

1. Among cereals, rice has the best protein quality from the point of view of lysine content and the amino-acid profile. However, the currently grown rice varieties have a low protein quantity, ranging only from about 5 - 10%. Indicavarieties usually have a little higher protein content than japonica strains. Also, the concentration of proteins in the aleurone layer leads to a considerable loss of proteins during milling. Any improve- , ment made in the quantity and quality of proteins in rice will be a significant contribution towards bridging the rapidly widening gap between the supply and requirement of proteins in the world. 2. The variability found in natural populations is usually the result of selection, natural and human, acting upon random mutations and recombinations. Protein quantity and amino-acid composition are new para- meters in human selection. Hence, the natural variability for such characters is not likely to be wide, as is already indicated by the screening for lysine carried out at the International Rice Research Institute. How- ever, where useful natural sources of high protein and high lysine content occur, these should be utilized in breeding. It would be useful if the Joint FAO/IAEA Division received from the IRRI a list of promising high protein and high lysine cultures as soon as they are detected at the IRRI so that a list could be circulated among the participants and other rice mutation breeders. 3. Mutation breeding offers the greatest scope where the selection criterion used represents a new need by world agriculture. The induction of mutations in high yielding and photo-insensitive rice strains for protein quantity and quality may, therefore, lead to very beneficial results as is already clear from the preliminary work done in Japan and India. Such work can be undertakein at centres where competent biochemical help and facilities are available for screening large populations for lysine and protein. 4. Mutation breeding for protein quantity and quality should be carried out without sacrificing yield. For the immediate future it would be worth- while to concentrate on improving protein quantity without any adverse alteration of the amino-acid composition. RECOMMENDATIONS 119

5. Experiments should be started on assessingthe scope for a mutational alteration of protein distribution in the rice grain. It would be very valuable if the protein gets distributed in the entire endosperm rather than remains largely restricted to the aleurone layer and embryo. 6. It would be useful if data on the interaction between variety and location and season, with reference to protein characteristics, could be collected for the important rice varieties. ' This aspect could be taken into consideration by the International Rice Research Institute, which obtains information on yielding performance of the same varieties from many locations in South East Asia. The IBP adaptation trials in rice can also be used for this purpose. It would be desirable if information could be obtained on the interaction between variety, fertilizer application, placement and time. 7. The Rice Mutation Group congratulates the Joint FAO/IAEA Division for taking the initiative to organize a Panel Meeting in Sweden, in June 1968, on new approaches to breeding for better plant proteins. The Pro- ceedings of this Panel would greatly stimulate and aid the genetic up- grading of protein quality and enhancement of total protein production. 8. Mutation breeding has already proved to be an efficient and speedy method of changing the physical and chemical properties of starch, such as amylose content, gela'tinization temperature, and hardness of endosperm. Therefore, even good japonica varieties can now be made use of in indica areas after the alteration of the properties of starch, and vice versa. This has become an excellent substitute for the japonica X indica hybridization program sponsored by FAO. The high yielding indica variety IR-8 can be made more acceptable through the mutational rectification of its grain quality.

~~LIST OF PARTICIPANTS~~

~~CO-OPERATORS~~

~~ANDO, A. Agricultural College, University of Sao Paulo, Piracicaba, S.P., Brazil~~

~~GANASHAN, P. Agricultural Research Station, Maha-Illuppallama, Ceylon~~

~~HAQ, M.S. Pakistan Atomic Energy Commission, Atomic Energy Centre, P.O. Box 164, Ramna, Dacca, East Pakistan~~

~~HU, C.H. Genetics Laboratory, Department of Agronomy, College of Agriculture, Taiwan Provincial Chung-Hsing University, Taichung, Taiwan, Republic of China~~

~~KHAMBANONDA, P. Breeding Division, Rice Department, Ministry of Agriculture, Bangkok, Thailand~~

~~MIAH, A.J. Plant Genetics Section, Atomic Energy Agricultural Research Centre, Tandojam, West Pakistan~~

~~REE, J.H. Yungnam Crop Experiment Station, Office of Rural Development, .Milyang, Korea~~

~~SWAMINATHAN, M.S. Indian Agricultural Research Institute, New Delhi-12, India~~

~~TANAKA, S. Institute of Radiation Breeding, Ministry of Agriculture and Forestry, Ohmiya, Ibaraki-ken, Japan~~

~~VIADO, G.B. Philippine Atomic Energy Commission, 727 Herran Street, Manila, Philippines~~

~~121 122 LIST OF PARTICIPANTS~~

~~PARTICIPANTS~~

~~AKEMINE, H. Department of Physiology and Genetics, National Institute of Agricultural Sciences,. Nishigahara, Kita-ku, Tokyo, Japan~~

~~FUTSUHARA, Y. Faculty of Agriculture, Nagoya University, Furo-cho, Chigusa-ku, Nagoya, Japan~~

~~HARN, H. Radiation Agricultural Research Institute, Seoul, Korea~~

~~ISHIKURA, H. Agriculture, Forestry and Fisheries Research Council, Ministry of Agriculture, Kasumigaseki, Chiyoda-ku, Tokyo, Japan~~

~~ITO, R. National Central Agricultural Experi- ment Station, Konosu 1227, Saitama-ken, Japan~~

~~KAWARA, K. Koyanagi-cho 3-11-30, Fuchu, Tokyo, Japan~~

~~LE TRUNG LAP Vietnam Embassy, Moto-Yoyogi 50, Tokyo, Japan~~

~~MARIE, R. Centre de Recherches Agronomiques du Midi, Station d'Amelioration des Plantes, Montpellier (Herault), France~~

~~MATSUO, T. Faculty of Agriculture, Tokyo University, Yayoi, Bunkyo-ku, Tokyo, Japan~~

~~MURAKAMI, K. Division of Genetics, National Institute of Agricultural Sciences, Hiratsuka, Kanagawa-ken, Japan~~

~~OGURA, T. Agriculture, Forestry and Fisheries Research Council, Ministry of Agriculture, Kasumigaseki, Chiyoda-ku, Tokyo, Japan 123 LIST OF PARTICIPANTS~~

~~OSONE, K. Faculty of Agriculture, Tokyo University, Yayoi, Bunkyo-ku, Tokyo, Japan~~

~~PARK, L.A.I. Yungnam Crop Experiment Station, Office of Rural Development, Milyang, Korea~~

~~RAJAN, S.S. Indian Agricultural Research Institute, New Delhi-12, India~~

~~SAKAI, K.I. National Institute of Genetics, Yata 1111, Mishima, Shizuoka-ken, Japan~~

~~Centro di Studi Nucleari della Casaccia, SCARASCIA-MUGNOZZA, G.T. C.N.E.N., Sa. Maria di Galeria, Rome, Italy~~

~~Department of Biology, SMITH, H.H. Brookhaven National Laboratory, Upton, L.I., New York 11973, United States of America~~

~~National Chugoku Agricultural Experi- TORIYAMA, K. Ment Station, Higashifukatsu 540, Fukuyama, Hiroshima-ken, Japan~~

~~School of Biological Sciences, VOHRA, F.C. University of Malaya, Kuala Lumpur, Malaysia~~

~~YAMASAKI, Y. Nihon University, Shimouma-cho 3-49, Setagaya-ku, Tokyo, Japan~~

~~INTERNATIONAL ORGANIZATIONS~~

~~JACKSON, R. Rockefeller Foundation, P.O. Box 2453, Bangkok, Thailand~~

~~KHUSH, G.S. International Rice Research Institute, Manila Hotel, Manila, Philippines~~

~~MIKAELSEN, K. Division of Research and Laboratories, International Atomic Energy Agency, 1010 Vienna, Austria 124 LIST OF PARTICIPANTS~~

~~YAMADA, N. FAO Regional Office for Asia and the Far East, Maliwan Mansion, Phra Atit Road, Bangkok, Thailand~~

~~SCIENTIFIC SECRETARIAT~~

~~SIGURBJORNSSON, B. Joint FAO/IAEA Division of Atomic Energy in Food and Agriculture, • International Atomic Energy Agency, 1010 Vienna, Austria~~

~~KAWAI, T. Division of Genetics, National Institute of Agricultural Sciences, Hiratsuka, Kanagawa-ken, Japan~~

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