Irradiation in successive generations: effects on dormant barley (Hordeum Distichum, L.) embryos

Radiation Botany, 1966, Vol. 6, pp. 535 to 543. Pergamon

Press Ltd. Printed

in Great Britain.

IRRADIATION IN SUCCESSIVE GENERATIONS: EFFECTS ON DISTICHUM, L.) EMBRYOS* DORMANT BARLEY (HORDEUM W. F. CAMPBELL? Crops Research Division, Agriculture Research Service, U.S. Department of Agriculture, Logan, Utah, U.S.A. (Receioed 7 July 1966) Abstract-Dormant seeds of barley (Ho~dcum distichum L.) were treated with a single acute exposure of ionizing irradiation applied once in each of three successive generations. Irradiation exposures, up to and including even the highest dose level (30,000 R), caused a maximum reduction of only 10 per cent on germination proper, i.e. the emergence of root, coleoptile, and shoot. After the second irradiated generation, seedling height usually decreased, then after the third generation it increased. The decrease may have resulted from a build up of genetic burdens (presumably chromosomal interchanges), followed by an elimination (involving haplontic and/or diplontic selection) of aberrant cells. Experimental results indicated that: (a) The Hannchen germ line appears to be capable of adapting to irradiation as an additional environmental stress and (b) use of recurrent irradiation in consecutive generations, to increase and retain the frequency of mutations in this diploid variety of barley does not appear feasible because of its high level of discrimination against induced mutations. R&mm&Des grains d’orge (Hordeurn distichum L.) en dormance ont CtC trait& par une dose aigue de radiations ionisante pendant trois generations successives. Ces irradiations, y compris la plus forte dose (30,000 R) ont provoqut une reduction maximale des proprietes germinatives, c’est-a-dire, l’apparition des racines, du coltoptile et de la plantule, de 10 pour cent. Aprb irradiation de la seconde generation, la hauteur des plantules decroit en general. Par contre, a la troisitme generation, elle est accrue. La dimunition de croissance peut provenir de la presence de lesions gtnttiques (probablement des interchangementschromosomiques) suivie d’une elimination (impliquant une selection haplontique et/au diplontique) des cellules aberrantes. Les resultats exptrimentaux indiquent que: (a) La lignee de cellules germinatives de la variete Hannchen parait capable d’adaptation a l’irradiation consider&e comme stimulus extrinseque. (b) L’utilisation d’irradiations aux generations consecutives en vue d’accrdtre et de conserver la frequence de mutations dans cette varittt d’orge diploide ne parait pas rtalisable en raison de son niveau elevt de discrimination des mutations. Zusammenfassung--Ruhende Samen von Gerste (Hordeum dirtichum L.) wurden einer einxigen, akuten ionisierenden Strahlung ausgesetxt, die in jeder von drei aufeinanderfolgenden Generationen einmal angewandt wurde. Die Bestrahlungen, ansteigend bis zur ho&ten Dosis von 30000 R, verursachten eine maximale Reduktion der eigentlichen * This investigation is a portion of a thesis submitted in partial ftilment of the Ph.D. degree in the Department of Botany and Plant Pathology, Michigan State University. t Present address: Plant Science Department, Utah State University, Logan, Utah 84321, U.S.A. 535

W. F. CAMPBELL

536

Keimungsrate urn nur IO%, d.h. das Austreihen von Wunel, Koleoptile und Spross. Nach Bestrahlung der zweiten Generation nahm gewohnlich die Hijhe der Keimlinge ab, nach Bestrahlung der dritten dagegen nahm sie zu. Die Abnahme mag darauf beruht haben, dass sich gene&he Defekte ergeben haben (wahrscheinlich reziproke Translokationen), denen die Eliminierung aberranter Zellen folgte (einschliesslich haplontischer und/oder diplontischer Selektion). Die experimentellen Ergebnisse legen folgende Schliisse nahe: (a) Die HannchenKeimlinie scheint fahig zu sein, sich an Strahlung als eine zusatzliche Umweltbelastung anzupassen und (6) Anwendung wiederholter Bestrahlung von aufeinanderfolgenden Generationen, urn die Mutationshgufigkeit in dieser diploidenvarietat der Gerste zu erhohen und zu erhalten, scheint nicht durchftuhrbar, da sie induzierten Mutationen stark entgegenwirkt.

INTRODUCTION on biological effects of irradiation indicates considerable interest m the problemofradio-resistance (acquiredorinduced) of plants, as well as animals to ionzing irradiation. In plants, for example, ABRAMS and FREY(~) studied the effects of acute irradiation on germination and seedling vigour in oats by irradiating the dry seeds once in each of three successive generations. They observed a reduction in germination and in the number of surviving seedlings but found an increase in CALDECOTT and vigour of those surviving. NORTH(~)have recorded an increase in chlorophyll mutations induced in a hexaploid oat variety each generation through 6 successive generations of re-irradiation. They also observed greater variability for plant height, panicle type, and maturity date following the second irradiated generation. YAMAGUCHI,(~~) using dry seeds of barley and rice, observed an increase in germination and fertility of the X-2 generation over the X-l following recurrent irradiation. MEWISSENet a1.(12*13) collected AndropogonJil$Xus seeds from an uraniferous soil with radioactivity of the superficial layers ranging from 10 to 50 times background, and from a non-uraniferous but otherwise similarly mineralized, soil. Following single acute X-ray exposures, seeds from the long-continued low-level irradiated population exhibited higher vitality. There appeared to be an enhancement of the biological potentialities rather than a degeneration. This investigation was initiated to: (a) evaluate the effects on the germ line of Hannchen barley of ionizing irradiation applied to each of three successive generations ofdormant embryos; (b) determine if Hannchen barley (a diploid

PERUSALof the literature

variety) possesses the potentialities for induced resistance or susceptibility following irradiation of dry seeds; (c) compare the responses with developing embryos of the same variety; and (d) compare the responses of this diploid variety with the results obtained by others working with dry see&.(lAl%l9) MATERIALS AND METHODS . Dormant embryos of matured dry seeds of barley (Hannchen distichum L., emend. Lam. cv. Hannchen, C.I. 531), obtained from L. W. MERICLE,were used (Fig. 1). Much information was already available concerning the normal development and radiosensitivity responses of this barley following one generation of treatment. Moreover, this line of barley has been used extensively(s*lD~ll) and its inherent stability has been established. Seeds of uniform size were attached to germination blotter disks, embryo side down, by means of double-gummed masking tape and equilibrated to 5 to 10 per cent moisture content by storing in a desiccator over anhydrous calcium chloride.@) The mounted grains, sealed in petri dishes, were enclosed within plastic bags and shipped from Michigan State University to Brookhaven National Laboratory for irradiation treatment. The treated groups received single, acute X-ray exposures of 4000, 7500, 12,500, 15,000 and 30,000 R each generation at a rate of 1000 R/min, as measured by a Victoreen Integron. Physical factors for irradiation were : 250 kVp, 30 mA, l-mm Al filter in addition to inherent filtration, and a 30-cm focal distance. Control seeds were handled in an identical manner except for the irradiation. Upon return from Brookhaven National Laboratory, the grains were germinated in

IRRADIATION

IN SUCCESSIVE

X1 FIG. 1. Diagram illustrating during dormancy

GENERATIONS

OF DORMANT

XI application of X-irradiation to Hannchen in each of three successive generations.

replicates by rolling 10 seedsper replicate in wet paper towelling. Prior to rolling, the grains were positioned equidistant along a straight line S-10 cm from the top of the towelling, with their long axes parallel and vertical. The rolls were placed upright in glassjars with the lower ends immersed in approximately 1 in. of distilled water, and then were placed under bell jars during seed germination. At the end of 5 days, data were collected on germination percentage, semi-lethality, coleoptile height, root length, shoot height, number of roots and any observed abnormalities. Following the observations made after 5 days, 30 seedlings per each treatment per each treated generation, selected at random, were transplanted in the greenhouse to 6-in. pots (5 seedlings/pot) and allowed to grow to maturity. Pots of plants of each generation were placed on the greenhouse bench in a completely randomized arrangement. Average temperatures in the greenhousewere 75°F during the day, and 60°F at night. To hasten flowering, supplemental, artificial lighting was usedto extend the day length to 20 hr. Supplemental nutrient solution wasadded every 10 days, beginning two weeks after transplanting until plants had flowered. Main heads, harvested when the moisture

BARLEY

537

X, barley embryos

content of the grains reached approximately 10 to 20 per cent, were used in the subsequently treated generation. At the time of harvest, survival to maturity and fertility data were taken. Measurements, to the nearest millimetre, were made at the junction of the shoot and root. The longest roots were measured as a matter of convenience sincepreliminary work showed that the total root length, average root length, and longest root length gave identical curves when plotted asper cent of control. Also measurement of the longest root reduced the handling time during observations and minimized the dehydration caused by air exposure. All seedling growth measurements,root number, and fertility data were analysed using the Student’s “t” test for significance of difference between means (Table 1). RESULTS Germination

Germinability at the different dose levels applied to dormant embryos following the first, second, and third generation of irradiation was very good (Fig. 2). In this study the dormant embryos of dry seedssustained a total exposure of 10 to 75 times that of the early proembryo D

CAMPBELL

5 Q 60

4.000 .-.7.500 0 ---l2.500 l

o ---l5;000

-------30.000

l

SO 0

I GENERATIONS

dormant embryos receiving different dose levels either did not differ significantly from the controls or were significantly lower (1 per cent level) each irradiated generation (Table 1). Variability resulting from the treatments remained rather uniform each generation, increasing only slightly over that of the controls, but there was an overall decrease in the average coleoptile height as the exposure level increased (Fig. 3). The average coleoptile height for the 30,000 R dose level exhibited a decrease after the second generation of irradiation followed by an increase in the third treated generation.

R R R R R

2

3

IRRADIATED

FIG. 2. Influence of X-irradiation on germination (irradiated as dry seed in each of three successive generations). stage of developing embryos,t5) yet the maximum depression in germinability was only 10 per cent over that of the controls.

SEEDLING RESPONSES Coleofitile height The mean coleoptile

Table

heights

of seedlings

1. Percentage

significance

from

Shoot and root growth With one exception, irradiation at the various exposure levels reduced seedling growth significantly (Table 1). The growth depression showed a tendency to be directly correlated with the amount of irradiation applied with the roots generally being more sensitive than the shoots (Table 1, Fig. 5). Comparing the three generations, the shoot growth generally decreased in the second treated generation, but increased in the third treated generation (Fig. 4). The

of d$rence in meam, using Student’s Exposure

Generation

test

level ( x 1O8)

Criteria

4

7.5

12.5

15

30

-1 -1 -1

-1 -1 -1 -1 -1

-1 -1 -1 0 -1 -1 -1 -1 -1 -1

Coleoptile Shoot height Root length No. roots Fert. florets

-1 -1 -1 0 0

-1

-1 -1 -1 -1 -1

x-112

Coleoptile Shoot height Root length No. roots Fert. florets

0 -1 -1 0 -1

-1 -1 -1 0 0

-1 -1 -1 0 -1

-1 -1 -1 0 -1

X-1/2/3

Coleoptile Shoot height Root length No. roots Fert. florets

0 0 -1 0 0

0

-1 -1 -1 +1 -1

-1 -1 -1 0 -1

X-l

-,

“t”

less than that of the control;

-5

-5 -5 0 0

+, greater than that of the control.

-1

-1 -1 -1 -1

IRRADIATION

IN SUCCESSIVE

1

f

~1 ----15.000

0 -----34000 2

I GENERATIONS

OF DORMANT

BARLEY

539

$!sJq ____ ------ji ;2$ig1

SE

4.000 9 -.7.500 0 ---112.500 l

40

GENERATIONS

R R R R R

3

3

IRRADIATED

GENERATIONS

FIG. 3. Influence of X-irradiation on coleoptile height, expressed as percent of control (irradiated as dry seed in each of three successive generations).

IRRADIATED

FIG. 5. Influence of X-irradiation on root length, expressed as percent of control (irradiated as dry seed in each of three successive generations).

.Numberof roots This primary applied embryos roots in

1001

variety of barley normally has one root and 5 or 6 seminal roots. Irradiation at different dose levels to dormant significantly reduced the number of only a few instances (Table 1, Fig. 6).

In one case (12,500 R) there was a significant increase

j,

in the number

of roots over that of the

~“~~~..~/~~~ i 2 GENERATIONS

3 IRRADIATED

FIG. 4. Influence of X-irradiation on shoot height, expressed as percent of control (irradiated as dry seed in each of three successive generations). induced variability was greater in the treated groups than that observed for the controls. Except for the 4000 R dose level after the first generation, the induced variability following irradiation at 30,000 R was less than that for the lower exposure levels each treated generation,

and in one instance lessthan that of the control.

I*

f

SE

4,000 m-.7.500 *----12,500

6

l

50

z 4

L

o ---15.00b e -----?K&OOO .

R R R R R

2

I GENERATIONS

3

IRRADIATED

FIG. 6. Influence of X-irradiation on the number roots, expressed as percent of control (irradiated dry seed in each of three successive generations).

of a;S

540

W. F. CAMPBELL

controls. While the number of roots remained relatively constant in each generation, the induced variability increased slightly. At all dose levels, however, and for each irradiated generation, the induced variability was greater than that of the controls. Curiously for the first and third generations of irradiation, the variability induced by the 30,000 R dose level was less than that of the lower exposures.

fluctuation. The induced variability was, in all cases,greater than the variability in the controls and, in general, increased within a generation asthe level of irradiation increased. When spike fertility (expressed as per cent of control) was plotted against generation time (Fig. 8) only the 12,500R doselevel showedan increasewith each treated generation.

Swvi~ul to maturity Survival to maturity of plants from irradiated dry seed generally was related to the total level of irradiation applied to the seeds. Plants that survived to maturity, from seeds exposed to 30,000 R of X-rays, exhibited a large decrease after the second successive generation of irradiation followed by an equally large increase after the third generation (Fig. 7). The survival to maturity of plants from seeds exposed at the 7500 and 12,500 Rdose levels increased with each succeeding irradiated generation.

3 GENERAf10NS21RRADlAfED

FIG.8. Influence of X-irradiation on spike fertility, expressedaspercentof control (irradiated asdry seed in eachof three successive generations). SEEDLING

ABNORMAUTIES

Semi-lethality Plants having only coleoptiles, shoot, and/or roots, albinas, and those failing to survive to maturity in the greenhouse have been classified 50 as semi-lethals. The maximum semi-lethality 0 I 2 3 GENERATIONS IRRADIATED observed at the different doselevels increased as the level of irradiation increased (Table 2). FIG. 7. Influence of X-irradiation on survival to Semi-lethality values at the 4000 and 7500 R maturity (irradiated as dry seedin each of three exposures showed very good agreement. Those successive generations). at the 12,500 and 15,000R doselevels were near to one another. At the 30,000 R exposure, the Fertility maximum semi-lethality ranged from 11 to 18 The mean number of total florets per main per cent greater than that of all other doselevels. spike per plant was slightly higher than those of the controls. The fertility generally was directly Coleo~tile correlated with the level of irradiation applied The coleoptile-only (i.e. coleoptile present, but each generation. The spike fertility induced by no shoot) abnormality increasedin frequency in the individual dose level showed considerable each consecutively treated generation when the -.--c ---* ------

4.000 7.500 12.500 15.000 30.000

R R R R R

IRRADIATION

IN SUCCESSIVE

GENERATIONS

embryos were given 15,000 and 30,000 R of X-rays during their dormancy (Table 2). At the lower dose levels this mutation occurred only in the second and third irradiated generations. The appearance of the coleoptile-only mutation at the 4000,750O and 12,500 R dose levels, however, decreased in successively treated generations. It was entirely absent at the 4500 R exposure after the third generation of irradiation. Albina mutations Albina mutations, entirely absent in’ the control plants, occurred infrequently among the seedlings derived from irradiated dormant embryos. Following the second irradiated generation, albina mutations occurred in seedlings of embryos receiving 7500 and 12,500 R of X-rays (Table 2). After the third consecutively treated generation, albina mutants were recovered only from plants in the three highest exposure categories. The frequency of albinism was almost four times greater at the 15,000 and 30,000 R dose levels than at the 12,500 R exposure.

OF DORMANT

BARLEY

Other abnormalities

Other seedling abnormalities were of two types. First, a white stripe about 4 cells wide appeared near the margin of the 5th leaf of 1 seedling from dry seedexposed to 30,000 R of X-rays in the first generation. Second, the spike of 1 plant mutated from the normal 2 to 6-rowed barley, followed an exposure of 30,000 R to the dry seedin the third irradiated generation. The lateral florets, however, remained sterile. DISCUSSION

Measurements of coleoptile and shoot heights indicate the relative irradiation damage to the processesof cell division and cell elongation.04* 1~7) According to the data reported above, maximum root length and the number of roots are equally important criteria in measuring the effects of irradiation. Moreover, these data confirm earlier work with developing embryos of this samevariety of barley that showed roots to be more sensitive than shoots to irradiation.@@ 9~0) These data also indicate that root number

Table 2. Seedling abnormalities observedfollowing irradiation of embryos during their donnancy in each of three successivegenerations

Treatment

No. - seedlings

541

% semi-lethal

% coleopt. only

% albina

X-l Control 4000R 7500R 12,500R 15,000R 30,000R

200 188 278 188 263 267

o-00 0.53 2.88 744 646 7.13

o-00 o-00 0.00 0.00 0.76 0.37

0.00 o-00 o-00 0.00 o-00 0.00

X-112 Control 4000R 7500R 12,500R 15,000R 30,000R

189 181 137 189 140 148

o-00 4.42 3.64 3.70 5.00 21.60

0.00 1.06 0.72 1.59 0.76 0.75

oao o-00 0.73 2.28 o-00 0.00

X-l/2/3 Control 4000R 7500R 12,500R 15,000R 30,000R

200 112 208 106 188 197

o-00 0.00 0.96 2.83 Il.19 12.69

0.00 o-00 0.48 0.94 1.14 2.99

0.00 o-00 0.00 o-94 4.30 4.10

542

W. F.

CAMPBELL

is the criterion influenced the least by applying irradiation to dormant embryos, even in successive generations. This is further substantiated at a 50,000 R exposure level by MERICLE et al.(n) Thus it appears that, in dormant embryos of dry seeds, most of the roots or root primordia are already sufficiently formed that they can emerge by cell elongation. The most frequently observed effect in young seedlings in the current study was a reduction in the amount of growth as compared with controls at the end of five days (Table 1). However, the shoot heights and root lengths were rather variable within each treated generation. According to &LDECOrr et al.c4), a considerable range in seedling heights was associated with X-ray exposures at 10,000 and 20,000 R. In a recurrent-irradiation study, CALDECOTT and NORTH@) have observed that the variability of seedling heights increased noticeably over that of the controls following the second generation of irradiation and increased more with each successively treated generation. The fluctuations in seedling growth (shoot and root) make it difficult to draw any definite conclusions concerning the ability of these structures to adapt to recurrent exposures of ionizing irradiation. However, following the second successive generation of irradiation at the higher exposures, the shoot heights showed a large decrease followed by an equally large increase after the third treated generation. This phenomenon, the lower germination percentages, and the “coleoptile-only” anomaly, appear to substantiate a build up of genetic defects (presumably chromosomal interchanges) similar to that reported in oats by ABRAMS and FREY(~) following irradiation in successive generations. The recovery that followed the subsequent irradiated generation and the low incidence of albinism indicate that selection against dominant and recessive mutant cells is occurring.(6*‘) Whether this alternating cycle would continue with future subsequent irradiated generations remains to be determined. In this respect it does not appear that the shoots are any more sensitive to irradiation following the third generation than they were after the first. While ionizing irradiation introduces more variability into the population than that

exhibited by controls, the extent to which the degree of variability increases with increasing dose level is apparently limited. This conclusion seems warranted since both shoot height and maximum root length showed less variation after the dry seeds were exposed to 30,000 R than they did following 15,000 R and in a few instances even less than did the controls. The phenomenon is further substantiated as a “real” effect by the results obtained by MERICLE et af.(ln following irradiation of dormant barley embryos with 50,000 R. The reduced variability observed with high irradiation doses may be because the cells that suffer the greatest chromosomal damage at this dose level cannot compete with the normal cells and are thus prevented from making any further contributions. While there was a slight increase in the mean number of total florets per main spike per plant following irradiation of dry seeds over t$se of the controls, the fertility generally was directly correlated with an increase in the level of irradiation each generation. Hence, as was noted earlier by sARIC(“) the stimulative effect of irradiation for the production of extra yield of grain does not appear profitable at this time. These data contradict those of KUZIN@) who reported that a dose range of 750 to 1000 R of X-rays applied to seeds of rye increased the subsequent generation yield by 2 1 per cent. The Hannchen germ line appears to be capable of adapting to irradiation as an additional environmental stress. The adaptation seems to be accomplished by an alternating cycle, initially a build-up of the genetic burdens in one generation followed by their elimination in a subsequent irradiated generation. The data also seem to indicate that recurrent irradiation cannot profitably be used on successive generations to increase and retain the frequency of mutations in this diploid variety of barley, mainly because of its high level of discrimination against induced mutations.

Acktwwledgements-The author sincere gratitude to Dr. L. W. interest, encouragement, and the course of this investigation. greatly appreciated were Mr.

wishes to express his MERICLE

for

his deep

guidance throughout Others whose aid was L. A. kiAIRER of the

IRRADIATION

IN SUCCESSIVE

GENERATIONS

Biology Department, Brookhaven National Laboratory,Upton, New York for the irradiation and Miss HEIDI SCHLO~~ER for drawing Fig. 1. REFERENCES 1. ABRAMS R. and FREY K. J. (1958) Effect of recurrent X-radiation on germination and seedling vigor in oats. PTOC.Iowa Acad. Sci. 65,174-l 83. 2. CALDECOTT R. S. (1956) Ionizing radiations as a tool for plant breeders. In: Proc. Intern. Conf. Peaceful Uses At. Energy, Geneva 12,40-45, 1956. 3. CALDECO~~ R. S. and NORTH D. T. (1961) Factors modifying the radiosensitivity of seeds and the theoretical significance of the acute irradiation of successive generations. In: Mutation and Plant Breeding. NAS-NRC Publ. No. 891, pp. 365-404. 4. CALDECOTT R. S., FROLIK E. F. and Momus R. (1952) A comparison of the effects of X-rays and thermal neutrons on dormant seeds of barley. Proc. Natl. Acad. Sci. U.S. 38, 804809. CAMPBELL W. F. ( 1966) Irradiation in successive generations : Effects on developing barley (Hordeurn distichum, L.) embryos in situ. Radiation Botany 6, 525-534. EHRENBERG L., GUSTAFSSONA., LUNDQUIST U.and NYBOM N. (1953) Irradiation effects, seed soaking and oxygen pressure in barley. Hereditas 39, 493-504. GAUL H. (1961) Studies on diplontic selection after X-irradiation of barley seeds. Proc. Symp. of the Effects of Ionizing Radiations in See& and Its Significance for Crok Improvement. Karlsruhe, Germany, (1960) IAEA, Vienna. pp. 117-l 38. KUZIN A. M. (1956) The utilization of ionizing radiation in agriculture. In: Proc. Intern. Conf. Peaceful Uses At. Energy, Geneva 12, 149-156, 1956. 9. MER~CLF. L. W. and MERICLE R. P. (1957) Irradiation of developing plant embryos. I. Effects of external irradiation (X-rays) on barley embryogeny, germination and subsequent seedling development. Am. 3. Botany 44, 747-756.

OF DORMANT

BARLEY

543

10. MERICLE L. W. and MERICLE R. P. (1961) Radiosensitivity of the developing plant embryo. In: Fundamental Aspects of Radiosensitivity. Brookhaven Symp. Biol. 14, 263-286. Brookhaven National Laboratory, Upton, New York. 11, MERICLE R. P., MERICLE L. W. and MONTOOMERY D. J. (1966) Magnetic fields and ionizing radiation: Effects and interactions during germination and early seedling development. Radiation Botany 6, 111-127. 12. ME~I~~EN D. J., DAMBLON J. and BACQ Z. M. (1959) Comparative sensitivity to radiation of seeds from a wild plant grown on uraniferous and non-uraniferous soils. Nature 183, 1449. 13. MEWI~~EN D.J., DAMBLON J. and BACQ Z. M. (1960) Radiosensitivity of seeds of Andropogon coming from uraniferous and non-uraniferous soils of Katanga. Bull. Inst. Agron. et Sta. Rech. Gemblow. 1,33 l-338. 14. MIICAEL~EN K.and HALVOR~EN H.(1953)Experiments on the respiration of X-irradiated barley seeds. Physiol. Plantamm 6,873-879. 15 MOUTSCHEN J., BACQ Z.M.and HERVE A.(1956) ’ Action of X-rays on the growth of barley seedlings. Experientia 12, 314-315. SARIC M. R. (1958) The dependence of irradia16 tion effects in seed on the biological properties of the seed. PTOC.Intern. Conf. Peaceful Uses At. Energy, Geneva 27, 233-248, 1958. 17. SICARD M. A. and SCHWARTZ D. (1959) The effect of high doses of radiation on seedling growth. Radiation Res. 10, 1-5. 18. SWAMINATHAN M. S. (1961) Effect of diplontic selection of the frequency and spectrum of mutations induced in polyploids following seed irradiation. Proc. Symfiosium on the Effects of Ionizing Radiations on Seeds and Its SigniJicance for Crop Improvement. Karlsruhe, Germany, 1960. IAEA, Vienna, pp. 279-288. 19. YAGAMWHI H. (1962) Induction of mutations by repeated irradiations in barley and rice. Japan 3. Breeding 12, 141-147.