Radiobiological studies in plants. X. Mutation rate induced by ionizing radiations at the al locus of sand oats

Radiation Botany, 1964, Vol. 4, pp. 503 to 516. Pergamon Press Ltd. Printed in Great Britain.

R A D I O B I O L O G I C A L STUDIES IN PLANTS. X. M U T A T I O N RATE INDUCED BY I O N I Z I N G RADIATIONS AT T H E al LOCUS OF SAND OATS* ICHIZO NISHIYAMA, SADAO ICHIKAWA and ETSUO AMANO t Faculty of Agriculture, Kyoto University, J a p a n

(Received 25 November 1963) A b s t r a c t - - I n order to elucidate a relationship between radiation dose rate and somatic mutation ~'ate, a mutant line of sand oats known as green inconstant, heterozygous for plant color gene ( + / a l ) , was used in the present study. Dry seeds of the green inconstant were exposed to neutrons from a reactor and g a m m a rays from Co 6° or Cs:3L All of the doses were simultaneously measured with several appropriate dosimeters at every irradiation treatment (Tables 1 and 2). Distinct white stripes were found in leaf 3-6 of seedlings, but those in leaf 1 and 2 were too small and indistinct to score• All induced stripes in leaf 3-6 extended from the base to the apical part of leaf blades and remained the same width throughout. The number of white stripes per leaf and the average ratio (p %) of white stripe width to leaf width were used as criteria of the somatic mutation frequency by radiation treatments. It is evident that the frequencies of white stripes increase with dose in each leaf, and the dose-response relationship does not appear to be linear, especially in the gamma-ray treatments (Figs. 7 and 9). Dose-rate effects were also observed, the higher dose rate being more effective. These responses are explicable by assuming a combination of one-hlt and two-hit events. O n the other hand, the frequencies of white stripes was reduced successively from leaf 3 to 6 (Figs. 6 and 8). This phenomenon seems mainly to be due to the developmental pattern of plant, and partly to elimination of mutant cells. Based on a consideration Of leaf ontogeny it is finally concluded that the average ratio (p %) of white stripe width to leaf width in leaf 3 shows more approximate value of the mutation rate at the al locus than those in leaf 4 to 6 (Table 4). R~s~--Afin d'dlucider la relation existant entre la dose de radiation et le taux de mutations somatiques, une lign~e mutante d'avoine des sables, connue comme ~tant verte mais d'une mani~re inconstante et h&~rozygote pour le g~ne de couleur ( + / a l ) a fit~ utilisde dans la pr~sente &ude. Des semences s~ches de cette vari&d ont ~t~ expos~es aux neutrons d ' u n rfiacteur et ~t des rayons g a m m a du Co 6° ou Cs ~a~. Toutes les doses ont dt~ mesur&s avec diffdrents dosim&res ad~quats et apr& chaque irradiation (Tableaux 1 et 2). O n a observ~ des bandes blanches bien distinctes de la 3~me /~ la 6~me feuille de la plantule, mais celle de la l~re et de la 2~me feuilles dtaient trop petites et indistinctes pour fitre relev&s. Toutes les bandes induites dans les feuilles 3-6 s'~tendaient de la base ~ la partie apicale du limbe foliaire et eonservaient partout la m~me largeur. Le nombre de bandes blanches par feuille ainsi que le rapport moyen (p %) de la largeur d'une bande blanche ~t la largeur foliaire a dtd utilis~ comme crit~re en vue d'appr~cier la frdquence de mutations somatiques induites par irradiation. I1 est ~vident que dans ehaque feuille, les frdquenees *Contribution from the Laboratory of Genetics, Faculty of Agriculture, Kyoto University, J a p a n , No. 303. T h e present study was supported partly by a grant from the Scientific Research F u n d of the Ministry of Education. tPresent address: Biology Department, Brookhaven National Laboratory, Upton, L.I., N.Y., U.S.A. 503

504

RADIOBIOLOGICAL STUDIES IN PLANTS. X de bandes blanches s'accroissent avec la dose et que la relation dose-effet ne paralt pas 6tre lin,~aire spdeialement apr~s traitement par les rayons gamma (Figs. 7 et 9). On a aussi observ6 les effets du d6bit de dose. Les d~bits de dose 61ev6s sont les plus et:ficaces. Les effets s'expliquent en supposant une combinaison des 6v~nements monotopiques et ditopiques. D'un autre c6t6, les frdquences des bandes blanches 6t6ient successivement rdduites en allant de la 3,}me ~ la 66me feuille (Figs. 6 et 8). Ce ph6nom~ne paralt principalement ~tre dCa au mode de ddveloppement de la plante et partiellement ~ l'dlimination des cellules mutantes. En se basant sur des consid6rations d'ontog6nie foliaire, on conclut gdndralement que le rapport moyen (p %) de la largeur des bandes blanches ~ la largeur foliaire de la 36me feuille indique une valeur plus approch6e du taux de mutations du lieu al que celles des feuilles 4/~ 6.

Zusammenfassung--Ziel der vorliegenden Untersuchung war, die Beziehung zwischen Strahlendosisrate und somatiseher Mutationsrate zu kl~iren. Hierfiir wurde eine mutierte Linie von Sandhafer, die als grtin-inkoustant, d.h.heterozygot fiir das Farbgen (+/al), bekannt war, benutzt. Trockene Samen der griin-inkonstanten Linie wurden mit Neutronen eines Reaktors und mit Gammastrahlen ~ n e r Co e° oder Cs T M .Quelle bestrahlt. Alle Dosen wurden w~ihrend der Applikation mit einer Reihe geeigneter Dosimeter gemessen (Tabelle 1 und 2). Im Blatt 3 his 6 der Keimlinge zeigten sich deutliche weisse Streifen. In Blatt 1 und 2 waren sic zu schmal und undeutlich ftir eine Analyse. Alle in Blatt 3 bis 6 induzierten,Streifen reichten von der Basis bis zum apikalen Teil der Blattspreite und hatten durchgehend die selbe Breite. Die Zahl yon weissen Streifen pro Blatt und das durchschnittliche Verh~iltnis der Breite der weissen Streifen zur Breite des Blattes in Prozent wurden als Kriterium ftir die somatische Mutationsh~iufigkeit nach Bestrahlung benutzt. Es l~isst sich zeigen, dass die H~iufigkeit der weissen Streifen je Blatt mit der Dosis zunimmt. Die Korrelation mit der Dosis scheint jedoch nicht linear zu sein, besonders nach Gammabestrahlung (Fig. 7 und 9). Eine Wirkung der Dosisrate liess sich ebenfalls beobachten, und zwar war die jeweils h6here Dosisrate auch wirksamer. Diese Reaktionen lassen sich erkl~iren, wenn man eine Kombination von Ein-Treffer und Zwei-Treffervorg~ingen annimmt. Auf der anderen Seite nahm die H~iufigkeit weisser Streifen von Blatt 3 his 6 schrittweise ab (Fig. 6 und 8). Diese Erscheinung diirfte vor allem auf die Entwicklungseigenarten der Pflanzen, zum Teil auch auf die Eliminierung mutierter Zellen zurtickgehen. Ausgehend von einer Betrachtung der Blatt-Ontogenese wird abschliessend festgestellt, dass das durchsehnittliche prozentuale Verh~iltnis der Breite der weissen Streifen zur Blattbreite bei Blatt 3 einen genaueren N~iherungswert ftir die Mutationsrate im al-loeus liefert, als bei Blatt 4 bis 6 (Tabelle 4).

1. INTRODUCTION MUTAGENIC effects of ionizing r a d i a t i o n s have been d e m o n s t r a t e d in m a n y papers i n the field of p l a n t genetics. However, i n most of t h e m the m u t a t i o n rate was estimated indirectly, e.g., b y scoring the frequency of m u t a n t s i n the X , generation. A n a d v a n t a g e o u s way to d e t e r m i n e the m u t a t i o n rate in the X 1 g e n e r a t i o n is to score directly the i n d u c t i o n of somatic m u t a tions i n plants heterozygous for various color genes. T h e first a t t e m p t was u n d e r t a k e n by SPARROW a n d POND(~6) who treated a clonal line of Antirrhinum heterozygous for a flower color gene with g a m m a rays. T h e y a n d their

co-workers m a d e further studies o n r a d i a t i o n i n d u c e d m u t a t i o n s in Antirrhinum heterozygous for three flower color genes, observing the n u m b e r a n d size of i n d u c e d m u t a n t sectors in petals.(4,s,~2,~4, 9-5) STEIN a n d STEFFENSEN(27'28), LATTERELL(XS), NATARAJAN a n d MARIe(15) a n d SMITH et al.(~s) i r r a d i a t e d maize seeds h a v i n g a genotype Yg2]yg, for p l a n t color, a n d c o u n t e d the average n u m b e r of y e l l o w - g r e e n sectors per leaf as a criterion ass~sing ,the a m o u n t of genetic damages. DAVIES"a n d WALL(6,7) iased a clone of Trifolium heterozygous for a leaf color. Besides, SPARROW et al.(~) reported somatic m u t a t i o n rates of flower color genes in Liliurn,

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FIG. 1. W h i t e stripes on the leaves of " g r e e n i n c o n s t a n t " oats after radiation treatment, st shows white stripe.

R.B.f.p.

5 O l.

ICHIZO NISHIYAMA, SADAO ICHIKAWA and ETSUO AMANO

Tradescantia, and Petunia, SAND et al.(*t) in Wicotiana, ALSTON and SPARROW(l) in Impatiens, SI-IAVER and SPARROW(2~) in Tropaelum, Phaseolus, and Tulipa, and Nzzu(l e) in Tulipa.

505

employed..After irradiation they were sown in wooden flats filled with steamed soil. Seed germination, seedling growth, and occurrence of color mutations in leaves were carefully T h e present study was undertaken to deter- obser~red. The results can be summarized briefly mine the dose-rate effect on the induction of as follows: somatic mutation in diploid oats heterozygous (1) Green and albinoseedlings segregated in for plant color following treatments with a ratio of 1:1 in the non-treated lot of green thermal neutrons and gamma rays. inconstants. However, the frequency of albino seedlings was clearly decreased with increasing doses of radiations. From this fact it is apparent 2o MATERIALS that "albino seeds" which give albino seedlings A mutant oat, "green inconstant" (2n=14), are more sensitive than "green seeds". which segregates green and albino plants in a (2) No color mutation in leaves was observed ratio of 1 : 1 and gives no homozygous greens, in'the non-irradiated lot 6f green inconstants or has been found in the progeny of triploid in irradiated A. strigosa. However, some seedhybrids between Arena barbata Pott. (2n=28) tings from irradiated green inconstants had and A. strigosa Schreb. (2n=14).o~,ts) Its geno- white stripes in the leaves. T h a t is, in leaf 1 typic constitution is represented by the formula, and 2 there were found many stripes or spots al+ + [ + R e X; al being a gene for albino, Re which were usually too small and indistinct to for early ripening and X for zygotic mortality, be scored, accurately. In leaf 3-6 of primary and no recombination is found among these culms, on the other hand, distinct white stripes genes. In this strain we easily get many thousand appeared from the base to apex of leaves. T h e of seeds heterozygous for albino without any white stripes showed several different widths, procedure such as artificial crossing. Only seeds but each one usually kept the same width along from the first florets of spikelets were used in the the full length of the leaf blade except for the present experiment, because of their uniform apical part, and generally on both surfaces," size. Seeds ofA. strigosa were employed as control upper and lower, of leaf blades (Fig. 1). From plants. these observations it is apparent that in mature seeds young leaf 1 and 2 had been formed, 3. E X P E R I M E N T I while only Primordia or masses of initial cells A preliminary experiment was carried out to leaf 3 and probably those to leaf 4 had been from J a n u a r y to March, 1961, in order to slightly differentiated. This ,consideration is determine adequate dosages of radiations for confirmed by the facts obtained from the histoinduction of somatic mutation in oat leaves. logical investigations of oat seeds by BONNETT(2) The dry seeds were sealed in small polyethylene and NISI~IYAMAet al. (unpublished). bags and exposed to thermal neutrons or X-rays. (3) The frequency of white stripes per leaf The thermal neutron treatment was performed blade increased with an increase of radiation in the beam hole No. 7 of JRR-1 (Japan doses (Fig. 9). On the other hand, another Research Reactor No. 1 at Tokai)0°, ~0) and index of mutation induction was the deterthree different doses, 3.1, 5.3, and 6-8 x 1012nth/ mination of an average ratio (p%) of white cm 2, were applied with a dose rate of about stripe width to leaf width which was calculated 2.1x10SntJcm*/sec. In this beam hole, how- by the following formula: ever, thermal neutrons were contaminated with l n 2~)1 p (%) = - z - - . 100 fast neutrons, resonance neutrons and gamma h i = 1 WI rays (see N m I ~ r v ~ et al.(lg,2o)). The X-ray treatments with three different doses, 7.5, 10, where p i s the average ratio (%) ofwhit6 stripe and 15 kr, were done with a dose rate of 362 width to leaf width, n is the number of leaves observed, wi is the width of white stripe in the i-th r[min, In each experiment lot 250-500 seeds were leaf of the same leaf order, and Wi ia the width of

RADIOBIOLOGICAL STUDIES IN PLANTS. X

506

the i-th leaf excluding the vein width, because cells of veins are colorless. This criterion represents that what percent of leaf width is occupied by white stripes as an average of all the leaves of a leaf order. Usefulness of this criterion as an index of mutation rate will be easily understood by the following diagram:

At the time of

measurement

,

At the time of irradiation

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namely, if (a) only four or (b) eight cells at the time of irradiation later form a leaf blade, occurrence of mutation at al locus in one cell will bring about one white stripe occupying (a) one fourth or (b) one eighth of leaf width

respectively, unless there is any competition between mutant and normal cells. Thus the average ratio (p %) of white stripe width to leaf width calculated from above formula will represent an average value of somatic mutation rate. I f there were no selection against mutant cells, the same p value would be obtained in leaf 3-6, but if there is selection, the p value must decrease from leaf 3-6, and it represents an underestimated value of somatic mutation rate. The results in Experiment l are presented in Fig. 3. The average ratio of white stripe width to leaf width, as well as the number of white stripes per leaf, decreased from leaf 3 to 6 (Figs. 2 and 3). This phenomenon can be easily understood by assuming that the leaves are in sequential stages of development at the time of irradiation and that certain of m u t a n t cells fail to continue dividing. ° From the preliminary investigation, it is apparent that the green inconstant has merit as a plant in which the induction of somatic mutations at the al locus can be studied. This mutant oat appears to have the additional advantage of providing some important information regarding organ ontogeny in plants•

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ICHIZO NISHIYAMA, SADAO ICHIKAWA and ETSUO AMANO .30

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/ Fro. 3. Average ratio (p o,e) of white stripe width to leaf width (leaf 3-6) in Experiment 1.

4. E X P E R I M E N T 2 ployed. The seeds of each lot were divided into A second experiment was performed from five, ten, or fifteen groups of thirty seeds, and October to December, 1961, to investigate the were randomly sown in thirty-two wooden flats relationship between mutation rate and dose filled with steamed soil and placed in a greenrate, and to study the effects of different kinds of house. radiation. Thermal neutron irradiation was con- Germination rate and seedling growth ducted in hole 7 when the reactor, JRR-I/10, 9.o) The germination rate on seventeenth day was operated at 40 kW and also in hole 16 at after sowing decreased with increasing doses of 0.5, 5, and 4 0 k W . Radiation doses were thermal neutrons or gamma rays, but it seemed measured by gold foils for neutrons, and by to be independent of dose rate used (Fig. 4). silver-activated phosphate glass dosimeters(ZX,x~) Similar relationships could be represented in for neutrons and contaminating g a m m a rays, plant height of 17-day-old green and albino each time the seeds were exposed. The doses and seedlings (Fig. 5). These responses appear to be dose rates applied in the experiment are sum- non-linear. G a m m a rays from Cs z87 seemed to marized in Table 1. The gamma-ray irradiation be more effective than those from Co s° on the was performed at 10 kc Coe°-facility(8) in the inhibition in seed germination and seedling J a p a n Atomic Energy Research Institute, Tokai, growth. Although the similar phenomenon was and 6 kc CsZS~-facility in the National Institute also observed on somatic mutation rate as seen of Genetics, Misima. Gamma-ray doses and in Figs. 7 and 9, it is hard to say that the dose rates were measured by Fricke dosimeters different effects were specific for gamma rays at the time the seeds were exposed. The results from two different sources, because those seeds are given in Table 2. exposed to Co 6° gamma rays were sealed in In each lot 300 or 450 seeds of the green glass containers while those exposed to Cs T M inconstant or 150 seeds of A. strigosa were era- gamma rays were covered with an aeryl-resin

508

KADIOBIOLOGICAL

STUDIES

IN PLANTS.

X

Table 1. A list of radiation doses applied in each experiment lot in aTRR-1. Thermal neutron fluxes and doses measured by gold foils and contaminating gamma-ray doses by glass dosimeters Lot No.

2 3 4 5 6 7 8 9 10 13 2S 13S++

Beam hole No.

Material

g r e e n inconst. . . . . . . . . . . . . . . . . . . . . . ,,

Power of reactor (kW)

.

16 . .

.

.

.

.

.

.

40

. .

0'~5

7' 16 7

A. stri. ,,

(nth/cm2/sec) * 4.47 x 4.30 4.51 5.61 x 5.53 5.74 6.87 x 6"58 5.59 2.12 x 4.23 x 2.12 x

~

. .

Thermal n e u t r o n flux

4'6 ,, ,,

1011 ,, ,, 101° ,, ,, 10 ~ ,, ,, l0 s 1011 l0 s

Exposure time (see)

Thermal n e u t r o n dose (nth/cm~) *

Contaminating gamma-ray d o s e (r)~

8.1 14-2 21.6 56 112 168 560 1120 1680 17940 7.5 17940

3.62 x 1012 6.11 ,, 9.74 ,, 3.14 ,, 6.20 ,, 9.64 ,, 3.85 ,, 7.37 ,, 9-40 ,, 3.80 ,, 3.17 ,, 3"80 ,,

908 1530 2240 785 1550 2410 963 1840 2350 5670 793 5670

*Including resonance neutrons. t C a l c u l a t e d f r o m t h e a v e r a g e c o n t a m i n a t i o n ratios, 1 r/6.7 x lOSnth.cm -2 a n d 1 r/4.0 x lO°nth.em -2 o b t a i n e d b y glass d o s i m e t e r in h o l e 7 a n d 16 respectively. + I r r a d i a t e d t o g e t h e r w i t h L o t 13.

Table 2. A list of dose rates and doses of gamma rays applied in each experiment lot, measured by Fricke dosimeters Lot No. 21 22 23 24 25 26 17 18 19 27 28 29 21S 29S*

Material g r e e n inconst. . . . . . . ,, . . . . . . , , . . . . . . . . .

Source C o 6° . . ,, . . Cs187 ,. . . .

~

yy

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C o 6° Cs tS~

,,

* I r r a d i a t e d t o g e t h e r w i t h L o t 29.

Dose rate (r/min) 29200 27000 25300 315 298 303 51-0 53"5 54"0 4.33 4.67 4"78 29600 4.78

Exposure time (see) 14"9 29"8 44.7 1200 2400 3600 6000 12000 18000 54000 108000 162000 14.8 162000

Dose (kr) 7.3 13"4 18"8 6"3 11.9 18"2 5" 1 10.7 16"2 3"9 8"4 12"9 7.3 12.9

I C H I Z O NISHIYAMA, SADAO I C H I K A W A and ETSUO AMANO plate. This relationship will be clarified in further studies. Dose-rate effects on the occurrence of white stripes As s t a t e d a b o v e i n E x p e r i m e n t 1,

irradiation of seeds with both X-rays and neutrons produces distinct white stripes in leaf 3-6 i n seedlings of green inconstants (Fig. 1). A n u m b e r of stripes appeared on only one

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FIG. 4. Germination rate of the seeds irradiated with thermal neutrons or gamma rays (on 17th day after sowing).

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FIG. 5. Seedling height of greens and albinos after irradiation with thermal neutror~ or gamma rays (on 17th day after sowing).

R A D I O B I O L O G I C A L STUDIES IN PLANTS. X

51(J

Table 3. Frequencies of white stripes appeared on either upper or lower surface of leaves

Induced by gamma rays

Induced by thermal neutrons Leaf order

Total no. stripes observed

No. stripes on one surface

%

Total no. stripes observed

No. stripes on one surface

%

Leaf 3 Leaf 4 Leaf 5 Leaf6

382 154 108 31

89 25 20 2

23.3 16.2 18.5 6.5

158 72 32 14

77 17 5 1

48.7 23.6 15.6 7.1

surface of leaf blades. T h e frequency of such single-surface stripes was least in the uppermost leaf (leaf 6) a n d greatest in the lowest leaf (leaf 3) (Table 3). A n d it was also seen that such single-surface stripes were more frequently induced by g a m m a - r a y treatments t h a n neutron treatments, especially in leaf 3 and 4. This p h e n o m e n o n should be certified in detail in further studies. A n average n u m b e r of white stripes, including single-surface ones, per leaf is calculated on leaf 3-6 as shown in Fig. 6. F r o m this figure it is

,

,

-

,

seen that the frequency of stripes per leaf exhibits a successive decrease from leaf to leaf up to leaf 6. This fact is explicable by the different cell numbers of leaf primordia at the time of irradiation, and besides by an assumption that the elimination of mtttant cells occurred in the course of development of plant tissue or organ, due to (1) the functional reduction in cell division or (2) developmental mechanism of the initial meristem in which a m u t a n t cell or cells were located. Considering that every white stripe resulted

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FIG. 6. Average number of white stripes per leaf (leaf 3-6) after treatments with 5.535.74 x 10t°nth/cm=/sec of thermal neutrons (hole 16, 5 kW) and 298-315 r/min of gamma rays ((3060).

I C H I Z O N I S H I Y A M A , SADAO I C H I K A W A and ETSUO 'AMANO

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Fxo. 7. Average number of white stripes per leaf (leaf 4) after treatments with various dose rates of thermal neutrons and gamma rays.

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FIO. 8. Average ratio (p %) of white stripe width to leaf width (leaf 3-6) after treatments = with 5.53-5"74 x 10 10 ntj,]em ]see of thermal neutrons (hole 16, 5 kW) and 298-315 r/min of gamma rays (Coe°).

512

RADIOBIOLOGICAL STUDIES IN PLANTS. X

from a single mutation event in a single cell (or on a single chromosome), the values for the occurrence of stripes could be used to determine the somatic mutation rate. However, frequencies of stripes per leaf appear to show more or less disagreement with a linear relationship corresponding with radiation doses, especially in the gamma-ray treatment (Figs. 6 and 7). In general, the higher dose rates were more efficient than the lower ones (Fig. 7). This fact can suggest that white stripes are induced by a combination of one-hit and two-hit events. On the other hand, an average ratio of white stripe width to leaf width is calculated from a formula modified from that used in Experiment 1 : 1 " wui+wl~ ' P (%) - - - -nY i=i

2w,

.100

where wui is the width of white stripe measured on the upper surface of the i-th leaf, wl, is that measured on the lower surface of the i-th leaf, and the other symbols are the same as those in the formula in Experiment 1, This modification was made because of the occasion~l occurrence of single-surface stripes. F i g u r e s 8 a n d 9 represent curves of p values calculated in each irradiation lot, and correspond to Figs 6 and 7 respectively. The results based on this criterion are very similar to those based on the number of white stripes per leaf. From Fig. 8 it is evident that the selection against mutant cells occurred in t h e course of development of leaves. In the course of expansion of leaf blade, however, no special selection against mutant cells seems to occur, because of no change of width of stripes from the base to apex of leaf blades. As discussed later, among the p values in leaf 3-6, that in leaf 3 is the most approximate value of the induced mutation rate in leaf primordia. Based upon this consideration, the induced rate of mutation at the al locus per unit dose of both thermal neutrons and gamma rays can be calculated from the p value in leaf 3 (Table 4). 4 × 108ntJcm * of thermal neutrons is estimated to be roughly equivalent to 1 r of gamma rays.

5.

DI$CUSSlON

In general, the number of white stripes per leaf, or the average ratio of white stripe width to leaf width increases with increasing radiation doses (Figs. 6, 7, 8 and 9). But the frequency of white stripes does not show a good agreement with a linear relationship with the increase of radiation doses, especially with gamma-ray doses (the slight disagreement with a linear relationship in case of thermal neutrons may be due to contaminating gamma rays as presented in Table 1). Such a result was already found by SPARROW et a/.(24.25) who counted the number of mutant spots in petals of Antirrhinum majus heterozygous for flower color. It is generally accepted that such a non-linear increase of the mutant character should be due to deletion of chromosome segments including the dominant locus through two-hit events. On" the other hand, STEIN and STEFFENSEN(28} obtained in maize leaves an expected linear relationship between the number of yellow-green stripes and X-ray doses. This case was reasonably explained by one-hit events which deleted the dominant gene because the gene is located on chromosome 9 near the end of short arm. SPARROW et al.(~5) also reported a similar linear result in Antirrhinum majus treated under chronic g a m m a irradiation. Besides total dose, the difference in dose rates plays some effects on the occurrence of white stripes (Figs. 7 and 9). The ratio among neutron dose rates was about 1 : 30: 260: 2000 (Table 1), while the ratios between lower and higher dose rates of gamma rays from Co s° and Cs 137 were about 1:90 and 1 : 12 respectively (Table 2). Higher dose rates appear to be more effective than lower dose rates, especially in high doses, in both neutron and gamma-ray treatments. It is noted, however, that the dose-rate effects were not clear or reversed in a few cases (Figs. 7 and 9). Its cause is not apparent, and the ,phenomenon will be ascertained in further studies. From the comparison of the RBE values of various radiations there has been offered the same conclusion that some fraction of the radiation-induced mutations were brought through chromosome breakage events.~9,8,14)

I C H I Z O N I S H I Y A M A , S A D A O I C H I K A W A and E T S U O A M A N O t.5

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20

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F1o. 9. Average ratio (p %) of white stripe width to leaf width (leaf 4) after treatments with various dose rates of thermal neutrons and g a m m a rays.

Table 4. Somatic mutation rate per al locus per unit dose of radiation, calculated on leaf 3 (a) T h e r m a l neutrons Dose rate (nth/em2/sec) 4.30--4.51 x 1011 5.53-5.74 x 101° 5.59-6.87 x 10' 2.12 x 108

R a n g e of doses ( x 10"nth/cm ~)

Mutation rate per locus* ( x 10-']4 x 108n~h.cm -2)

Remark

3.62-9.74 3-14--9.64 3.85-9.40 3-80

9.06-13-48 4.03- 9.35 5-06-10.39 1"52

Hole 16, 40kW ,, 5 kW 0.5 kW Hole 7, 40 kW

R a n g e of doses (kr)

Mutation rate per locus*

Remark

(b) G a m m a ray Dose rate (r/min) 2"53-2"92 × 104 2"98-3"15 × 10 ~ 5"10-5"43 × 10 4"33--4"78

7"3-18"8 6"3-18"2 5"1-16"3 3"9-12"9

( x 10-'/r) 0"95-3"51 1"27-2"88 3"86-9"42 2"82-5"23

*Calculated from p values.

C o so ~187

514

RADIOBIOLOGIC,AL STUDIES IN PLANTS. X

As stated above leaf 3 is hardly recognized in the mature seed of sand oats by the histological investigation. However, its primordiurn, composed of some cells, has probably differentiated more or less in the meristematic tissue, and those of the upper leaves will initiate later in the meristem. By irradiation treatment some or a few ceils of leaf 3 primordium mutated and these mutant cells could form white stripes in the leaf. Each stripe might be initiated from one mutant cell, because simultaneous mutations of two or more adjacent cells occur very rarely. It seems probable that white stripes in the other upper leaves originate from the other mutant cells in the meristematic tissue, a part of which will take part in the development of the subsequent leaves or various organs. In the course of such a development each mutant cell divides successively and forms a block of mutant cells. Then, it could be expected that wider stripes would be formed in upper leaves than in lower ones. However the observed results indicate no such a tendency, but average relative widths of stripes are nearly the same (3.4-4.7%) in leaves in each leaf order (Table 5). Therefore there must be considerable selection against mutant cells in the course of formation of the leaf primordia. O n the other hand each white stripe kept the same width along the full length of the leaf. This result suggests that mutant cells seem to divide at approximately the same rate as nonmutant cells after the leaf primordium was formed. And it is apparent that the leaf primordium was not formed from only one initial cell in the meristem, but from a mass of some initial cells.

O n the "one to one assumption", that one stripe is formed from one initial mutant cell, the following two considerations are given: (1) based upon 0.55 per cent of the narrowest stripe width or 3.38 per cent of average stripe width, the leaf 3 primordium might be consisted of about 180 or 30 cells in mature seeds, respectively; (2) the widest stripe could be formed by divisions of an original mutant cell about 5-6 times as often as the narrowest stripe in each leaf order. O n the other hand, the authors observed that some seedlings which showed white stripes, and were grown in the field, gave rise to a number of tillers, some of which exhibited white stripes or sectors in the greater parts of the culm and leaf. This fact indicates a remarkable increase of mutant tissue in tillers. For understanding these phenomena, histological studies on the structure and development of the oat plants may be further needed. At any rate, however, the data lead to the assumption that a leaf primordium is formed from some initial cells in the meristem zone of shoot apex. The division of each initial cell of the primordium proceeds laterally only in a certain limited number to form the leaf breadth, but divides vigorously longitudinally. Based on these findings, it could be concluded that the average ratio (p %) of white stripe width to leaf-blade width in leaf 3 gives more approximate value of the mutation rate in the leaf primordia than those in upper leaves. Comparing somatic mutation rates calculated from p values in leaf 3, genetic responses at the al locus to dose rates and different kinds of ionizing radiations are clearly seen (Table 4).

Table 5. Averages and ranges of the relative width (%) of white stripe to leaf width Leaf order Leaf 3 Leaf 4 Leaf 5 Leaf 6

No. of white stripes observed 540 226 140 45

Relative width (%) of white stripe to leaf width Average

Range

3.382 3.950 3.707 4.729

0.55-23.64 0-47-39.22 0.56-26.04 0.55-25-27

I C H I Z O NISHIYAMA, SADAO I C H I K A W A and ETSUO AMANO

Acknowledgements--The authors wish to express their appreciations to the members of the JRR-1 and the Co s° Laboratories, the Japan Atomic Energy Research Institute, for help in neutron and gamma-ray irradiations. They also wish to thank Dr. S. KONDO and Mrs. H. ISHrWA of the National Institute of Genetics for helpful technical advices in gamma-ray treatment, and to Mrs. S. UEMATSU, Mr. F. MOTOYOSHXand Mr. N. INOMATAfor the assistance in the experiments. REFERENC,ES 1. ALSTON"R. E. and SPAm~ow A. H. (1962) Somatic mutation rates in double and triple heterozygous of Impatiens balsamina following chronic gamma irradiation. Radiation Botany 1, 229-232. 2. BONNETT O. T. (1961) The oat plant: Its histology and development. Ill. Agrie. Expt. Sta. Bull. 672, 112 p. 3. CALD~.COTTR. S. (1955) The effects of X-rays, 2-MeV electrons, thermal neutrons, and fast neutrons on dormant seeds of barley. Ann. N.T. Acad. Sd. 59, 514-535. 4. CUANYR. L., SPARROWA. H. and JAHN A. H. (1958) Spontaneous and radiation-induced somatic mutation rates in Antirrhinum, Petunia, Tradescantia, and Liliura (Abst.). Proe. l Oth Intern. Congr. Genet. 2, 62-63. 5. CUA~ R. L., SPARROW A. H. and POND V. (1958) Genetic response of Antirrhinura majus to acute and chronic plant irradiation. Zeit. Vererb. 89, 7-13. 6. DAvms D. R. and WALL E. T. (1960) Induced mutations at the Vtv locus of Trifolium repens. I. Effects of acute, chronic and fractionated doses of gamma radiation on induction of somatic mutations. Heredity 15, 1-15. 7. DAvms D. R. and WALL E. T. (1961) Induced mutations at the VOr locus of Trifolium repens. II. Reduction below the additive base line by fractionated doses of gamma radiation. Genetics 46, 787-798. 8. Dept. of Radioisotope Applications (1960) The design and construction of a 10,000 curie cobalt-60 gamma irradiationfo~ility (JAERI 6002). Publ. by the Japan Atomic Energy Research Institute. 9. EHRENBEROL. and NYBOMN. (1954) Ion density and biological effectiveness of radiations. Acta Agr. Scand. 4, 396-418. I0. JRR- 1 Operation Group (1958) Operating characteristics of07RR-I (JAERI 1003-E). Publ. by the Japan Atomic Energy Research Institute.

515

11. KONDO S. (1960) Silver-phosphate glass dosimeters and their application to X-ray dose measurements inside X-rayed mice and tc separate measurements of thermal neutron fluxe~ and gamma doses in the JRR-1 Reactor. Ann. Rept. Natl. Inst. Genet. 10, 155-157. 12. KONDO S. (1961) Simultaneous measurement ot thermal neutron fluxes and gamma contamination doses by silver-activated phosphate glass, pp. 491-496. In: Selected Topics in Radiation Dosimetry. Intemat. Atomic Energy Agency, Vienna. 13. LAT'rERELL R. L. (1959) Changes in radiosensitivity of maize chromosomes during seed germination (Abst.). Genetics 44, 521-522. 14. MATBUMURA S., K O N D O S. and MABUGH! T. (1963) Radiation genetics in wheat, VIII. The RBE of heavy particles from Bl°(aV,oc)Li~reaction for cytogenetic effects in Einkorn wheat. Radiation Botany 3, 29-40. 15. NATAPa~JANA. T. and MARIC M. N. (1961) The time-inteusity factors in dry seed irradiation. Radiation Botany 1, 1-9. 16. N~.zu M. (1962) The effect of radiation on tulip breeding. Gamma Field Symposia in Japan, No. 1, 43-49. 17. NXSHIY~,A I. (1934) The genetics and cytology of certain cereals. VI. Chromosome behavior and its bearing on inheritance in triploid Arena hybrids. Mem. Coll. Agr. Kyoto Univ. No. 32, 1-157. 18. NISHIYAMA I. (1941) Cytogenetical studies in Arena, IV. Distorted Mendelian ratios due to the differential fertilization. 07ap. J. Genet. 17, 247264. 19. NISmYAMA I., IeHUCAWAS. and MARUVAV,A T. (1962) Radiobiological studies in plants, VI. Effects of radiations from the nuclear reactor, JRR-1, on dry seeds of wheats and oats. Japan. 07. Breeding 12, 237-245.

20. NISmVAMA I., ICHmAWA S., U~.MATSU S. and AMANO E. (1964) Radiobiological studies in plants, IX. Further studies on the growth inhibition by radiations from the nuclear reactor, JRR-I. aTapan. 07. Breeding 14, 69-74. 21. SAND S. A., SPARROW A. H. and SmTH H. H. (1960) Chronic gamma irradiation effects on the mutable V and stable R loci in a clone of Nicotiana. Genetics 45, 289-308. 22. SHAV~-RD. L. and SPARROWA. H. (1962) The relationship between nuclear or chromosome volume and rate of radiation-induced somatic mutation in higher plants (Abst.). Genetics47, 984.

516

RADIOBIOLOGICAL STUDIES IN PLANTS. X

23. SMITHH. H., CURTISH. J., WOODLEYR. G. and ST~IN O. L. (1962) The deutron microbeam as a tool in botanical research. Radiation Botany 1, 255-268. 24. SPARROW A. H. and CUANY R. L. (1959) Radiation-induced somatic mutations in plants. Proc. Conf. on Radioactive Isotopes in Agr., U.S.A.E.C. Rept. No. TID-7578, 153-156. 25. SPARROW A. H., CUANY R. L., MIKSCnEJ. P. and SC~aRER L. A. (1961) Some factors affecting the responses of plants to acute and chronic radiation exposures. Radiation Botany 1, 10-34.

26. SPARROWA. H. and POND V. (1956) Some cytogenetic and morphogenetic effects of ionizing radiation on plants. Proc. Conf. on Radioactive Isotopes in Agric., U.S.A.E.C. Rept. TID-7512, 125-139. 27. STEIN 0 . L. and STEFFENSEN D. M. (1959) Radiation-induced genetic markers in the study of leaf growth in Z.ea. Am. 97. Botany 46, 485--489. 28. STEIN O. L. and ST~FFENSEND. M. (1959) The activity of X-rayed apical meristems: A genetic and morphogenetic analysis in yea mays. Zeit Vererb. 90, 483-502.