A correlation of seedling height and chromosomal damage in irradiated barley seeds

Radiation Botany, 1969, Vol. 9, pp. 1 to 14. Pergamon Press. Printed in Great Britain.

A C O R R E L A T I O N OF SEEDLING H E I G H T AND C H R O M O S O M A L DAMAGE IN I R R A D I A T E D BARLEY SEEDS* ALAN D. CONGER and HARLAN Q. STEVENSON Radiation Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, U.S.A. and Southern Connecticut State College, New Haven, Conn. 06515, U.S.A.

(Received 19 September 1968) A b s t r a c t - - T h e most commonly used measurement of radiation damage to seeds is seedling height, the mean height a lot of seeds attains at some time during exponential growth. If planted immediately post-irradiation, seeds of a dose lot give a normal height distribution, but if stored before planting give very abnormal and even bimodal height distributions. By within-seed comparisons of chromosome abnormality (from roots excised at 24-36 hr) with height (attained by 7-9 days) in irradiated barley seeds, it is shown that damage to height and to chromosomes are closely correlated, even within a treatment in which great heterogeneity occurs. The two effects have equal radiosensitivity, but different shoulders to their dose curves. Seedling height is not depressed until 25-30 per cent of the cells bear chromosomal abnormalities. The heterogeneity observed is not due to a between-seed heterogeneity in dose or in oxygen content, and probably not in moisture. These experiments show that the heterogeneity arises from factors that operate on post-irradiation (indirect) storage damage, but are without effect on during-irradiation damage (direct). R~suna~----Le moyen le plus commundment utilis6 d'dvaluer le dommage dO aux radiations dans les graines est la hauteur de la plantule c.a.d, la hauteur moyenne qu'atteint un lot de graines ~t un moment donn6 de la croissance exponentielle. Lorsqu'on plante les graines imm6diatement apr~s irradiation, la distribution des hauteurs est normale pour une dose dormde. Par contre, si les graines sont stockdes avant d'etre plantdes la distribution des hauteurs est tr~:s anormale et m6me bimodale. Par comparaison des anomalies chromosomiques ~tl'int6rieur des semis d'orge irradi6s (pour des racines excisdes ~t24--36 h) avec la hauteur des plantules (atteinte en 7-gjours), on a montr~ qu'il existe une forte corr6lation entre le dommage pour la hauteur et le dommage chromosomique, m6me pour des traitements qui montrent une forte hdt6rog6n6it6. Les deux effets ont la m6me radiosensibilitd mais des courbes, d'inflexions diff6rentes. La hauteur des plantules n'est pas diminude avant que 25-30 pour cent des cellules contiennent des anomalies chromosomiques. L'h6tdrog6ndit~ observde n'est pas due ~ des diff6rences entre grains pour la dose, le contenu en oxyg~ne et probablement pas de l'humidit6. Ces experiences montrent que l'hdt~rogdn6it6 provient de facteurs agissant pendant le stockage sur le dommage produit apr6s irradiation (indirect) mais est sans effet sur le dommage produit pendant l'irradiation (direct). *Supported in part by a US Atomic Energy Commission Grant No. AT-(40-I)-2579 and by a Public Health Service Research Career Award No. 5-K3CA-25,930.

ALAN D. CONGER and HARLAN Q. STEVENSON delivered at about room temperature, and increased storage time--also enhance or maximize the bimodality or distortion of the seedling height frequency distribution. Given the normal and not-normal distributions just shown, it remained to test if the seedling height criterion itself for some u n k n o w n reasons was unreliable under these conditions. We did so by measuring in the same seeds the chromosomal as well as seedling height damage, and their correlation. For this within-seed correlation of chromosome aberrations and seedling height, it was necessary to show that: (1) removal of a root(s) on day one did not

affect the subsequent 7-9 days height attained by the same seed, (2) the aberration frequency was substantially the same in the 4-7 roots from the same seed, and finally, (3) whether the sought for height-aberration correlation did obtain. T h e results to follow show that all three conditions were fulfilled. T o show that the removal (at 24-36 hr) of one or two seedling root tips of the 4 - 7 that generally grow from a seedling did not impair its subsequent growth, i.e., its seedling height at 7-9 days, a comparison was m a d e of the 8-day height of c u t - u n c u t pairs of seeds from the sam.e treatment and growing alternately in the same

FIos. la and lb. Frequency distributions, number of seedlings vs. seedling height (cm)s at 7 to 8 days of growth for variously treated seeds. i

15.

El .,--~ I

I

a ~

IO

i

i

"~El

,,

x

s

~ I

•

Ir;l,.l~

~

A

~'~"A.~

0

'\ []

r:l ",. /"

.....o."~,,~

•

or5"b/

/

o..... T........... o ..... $

i

~..

I

I0

15

2O

SEEDLING HEIGHT {cm]

Fig. la. Planted immediately post-irradiation. Vertical arrows indicate mean heights for each curve. All irradiated in air.

• O I-1 A

Seed % water

Dose, kR

2.8 10.2 3"8 7.3

None None 50 40

No. of seeds 50 I00 72 50

Mean ht., cm

Ht. % of control

14.5 13.5 5.9 4.6

100 100 49 34

IRRADIATED BARLEY SEEDS I~) l

t

I

i

m 10

[

I

I

E]

I

n

8

I\

I

•

\

I

I

0

i

i

,

I

I

i

i

I

J/

\

I i

~ ~ ,.

,

/

/"~

",,_

o

I t~. 2 ..........

I

/'~,

i

i\i

/ i

i\~ ,~ i ~, ," ! ~'. ./ i ~_." "~.."

/

'

i

~ ' o

i

~"......

I

.o ~'.... ".~ ....... -~ o ...... '...

,,o

,.o .' .......

"~'B'~'D~__\

I

o

I

0

! i

/ ! ~/ ~J X .~,D/I ,. A/ i

• ~.~

10 L

i

i

i

i i i i

~

0

~ IO I

f

i~ (n:26/

'

! i

I \ I \ I \ I '.

5

i

5

,,,

.-.6" -0'.'

/

/ -

~ , " ~ ~.

I

o ........................... o ............................ '1

~

'h0 ,~_.~

~6

-~

.........

SEEDLING HEIGHT (cm)

Fig. lb. Seeds stored post-irradiation and before planting. To avoid overlap, each curve is displaced 4 cm to the right of its predecessor. Seed % water

[] /x O

2.5 2.8 2"8

Gas irradstored

Stored, days

Dose, kR

No. of seeds

Air-Air Air-Air N,-N2 (10atm.) 0,-0, (2 atm.)

6 7 3

15 4 10

50 50 49

6.9 6.9 4.1

58 49 35

7

8

78

6.0

47

dish, one with a root cut off, the other uncut. The mean 8-day heights from a total of 256 cut-uncut pairs, compiled from 4 different treatments and involving doses from 5-50 kR, differed by only 3.1% (t = 0.724, P > 0 . 4 , not significant). In another comparison, the seeds from one experiment (4 different doses, total 486 seedlings) were grown half in one dish and uncut, half in another but with 2 roots cut off at 2436 hr. The 8-day mean heights of seeds from the cut and uncut dishes did not differ by as

Mean ht, cm

Ht., % of control

much as 1% (max, differences, 0.9 per cent; min., 0 per cent). It is fair to say removal from a seed of 1 or 2 roots at 24-36 hr did not affect the 7-9 day seedling height subsequently attained. Variation between roots of a single seedling was tested by comparing individually the value for per cent normal metaphases for 25 rootpairs from 25 different seedlings (compiled from 10 different treatments, doses 1-36 kR, total 4350 cells). The average difference for all 25 pair comparisons was only 7 per cent of the

8

ALAN D. CONGER and HARLAN Q. STEVENSON 100

u~ o. < ~"

75

:E

Z O

50

O z

~a L

m.

I

4.32

50

I00

I$0

SEEDLING HEIGHT,ON 8th DAY [mm]

Fio. 2. Correlation of seedling height with percent normal (aberration-free) metaphases, for pairs of roots from the same seed. Each pair of points is for two roots from the same seed. The data illustrates the betweenroots correlation, as well as the correlation of height with percent normal metaphases. Total: 22 pairs of roots; 50 metaphases analyzed in each root tip. Seeds: 2.8 per cent water content, " C o y-irradiated with 0, 2, and 5 kR in 1½ atm. 02; stored 20 days in 1½ atm. Oz. The line drawn is the least-squares fitted regression line: Y(ht., ram) = 4"32 + 1"683X(% normalmetas.)"SE of estimate = 29.0 mm. [] Control (unirradiated); O 2 kR; A 5 kR.

per cent w a t e r content), in nitrogen, air, or oxygen a n d p l a n t e d i m m e d i a t e l y , or stored (3-90 days) in nitrogen, air, or oxygen (1.5117 atmospheres). A total of 17 doses a n d 290 roots are involved in the e x p e r i m e n t s j u s t summarized. W e conclude that, even with considerable heterogeneity in a t r e a t m e n t lot as in the cases cited above, seedling height of a n i n d i v i d u a l seed is a true a n d valid i n d i c a t o r of h o w m u c h r a d i a t i o n d a m a g e that seed has suffered.

T h e correlation b e t w e e n seedling height a n d c h r o m o s o m a l d a m a g e can be m a d e on a p o p u lation average basis also, as is done in Fig. 4, where dose curves for height a n d c h r o m o s o m e n o r m a l c y from the same lots o f seed are compared. I n F i g u r e 4, the m e a n heights of the total s a m p l e (81-97 seeds/dose) is the same as for the cytological subsample. This was achieved b y a p p l y i n g a small correction to the cytological sample. T h e a v e r a g e 8 - d a y height for the total

I R R A D I A T E D BARLEY SEEDS 100

,

,

9 n,

•

°°

o

~

90

oo / , , o

70

o

< z

o

~

o8

X

60

X

¢o o

80

z~ A

0

A

0

A V

S0

X

0

o

0 z

X o

40

D.

30

2O

vX8 v/

%

10

X

W o

~Ov

o

V

-X--W,×, 20

i 40

I

I

I

t

60

i

80

SEEDLING

i

I

100

HEIGHT

I 120

i

I 140

150

(mm)

Fzo. 3. Correlation of seedling height with percent normal (aberration-free) metaphases in the root tips of the same seed. Each data point represents a single seed, which had percent normal metaphases measured in two roots excised at 24-36 hr, then its height measured subsequently at 8 days of growth. Totals: 89 seedlings, 178 roots; a minimum of 50 metaphases were analyzed for each root tip. Seeds: 2.8 per cent water content, ,(-rays doses 0-5 kR, in air; stored 20 days in air, grown 8 days. The line drawn in is the least-squares fitted regression line: Y(ht., mm) ----24" 1 + I" 153X(% normalmetaphases).SEof estimate = 17"3 mm. The cluster of square points along the top, from the unirradiated control seeds and which show no abnormalities illustrates the variability in control growth. [] 0 kR; © -= 1; A = 2; S = 3 ; ~ = 4 ; × = 5 k R .

cytological s a m p l e is 10-15 p e r cent h i g h e r t h a n for the total sample, s i m p l y because on the first d a y w h e n roots a r e r e m o v e d the slows t a r t i n g seeds have not y e t g r o w n e n o u g h to furnish roots, or s u b s e q u e n t l y height, to the cytological g r o u p , b u t do c o n t r i b u t e l a t e r to the 8 - d a y h e i g h t o f the total sample. T o correct for this, two to six seeds were e l i m i n a t e d p r o -

gressively s t a r t i n g with the tallest until the m e a n h e i g h t o f the cytological s u b s a m p l e was the same, a t each dose, as the total sample. W e feel justified in d o i n g this as we have a l r e a d y shown b y i n d i v i d u a l within-seed c o m p a r i s o n t h a t height a n d n o r m a l c y a r e c o r r e l a t e d (Figs. 2 a n d 3, a n d text). All seeds, even s u p r a l e t h a l l y i r r a d i a t e d ones, will a t t a i n a constant length

12

ALAN D. CONGER and HARLAN Q. STEVENSON

carefully gas-equilibrated experiments with vacuum, 03, or N 2. In these, the seeds were extensively evacuated and flushed, held in the gas for long times (up to 7 days before radiation), and oxygen content forced to the limits (vacuum or pure N~, and Oo at 70 atm. for 7 days) where radiosensitivity is independent of oxygen content--see Figs. lb, lc, ld and A,iethods. This leaves, of the most likely suspects, seed moisture content as a variable. In our experiments, moisture content was controlled by longtime constant-humidity storage, mostly over one year but a minimum of 2 months, and with frequent mixing of the seeds until constant seed weight was attained. Although our moisture-equilibration was very long and thorough, we are hesitant to say the variability is not due to heterogeneity in moisture content. With X- or y-rays, it is known that seed moisture content has a profound effect on radiation sensitivity on immediately planted(3.4.11.14. 23) but more especially on stored seeds,(8,12) alters radiosensitivity most steeply in the region of 112 per cent water, and is most pronounced at low moisture contents, ca. 2-3.5 per cent water.(8) Effects on bacterial spores are similar.(2s) These water levels are so close to the lower limit of dryness attainable, ca. 0.9 per cent, that it is quite possible inhomogeneities in water content could exist at the intracellular level even in seeds of the same overall water content. Another factor affecting heterogeneity of seedling height is the L E T of the radiation employed, and because the influence of L E T interacts with the other factors affecting heterogeneity-storage, dryness, and oxygen---it must be considered. A lot of seeds which gives heterogeneous height distribution with low L E T X- or y-radiation gives remarkably uniform results with high L E T (neutron) radiation, a fact first observed and pointed out by CALDECOTT(6) and since then amply confirmed.O.a4.19) This observation might at first glance implicate oxygen to the exclusion of other variables, for as is well known the oxygen effect is maximal with low L E T X- or y-rays but only very slight with fast neutrons. We have already given the reasons, however, why we feel that oxygen difference does not account for the heterogeneity in our experiments.

Seedling height heterogeneity is maximal with low L E T radiation on stored, dry, nongas treated (i.e., air at normal pressure) seeds at room temperature. With the low L E T radiation, the heterogeneity can be eliminated by planting immediately (dry or wet, gas treated or not), and if stored, by oxygenequilibration or moisture ( ~ c a . 10 per cent moisture). With high L E T fission neutron irradiation, however, normal height distributions are obtained independently of whether seeds are planted immediately or stored, dry or wet, and gas-equilibrated or not.(2,5) In general, then, we can say that the heterogeneity is characteristic of low L E T irradiated, stored seeds, and is dependent on their moisture and oxygen content. Furthermore, as will be discussed later, just those factors that prevent or eliminate heterogeneity--high L E T radiation, or with low L E T immediate planting, and if stored, wetness and gas-equilibration-also prevent or eliminate development of aftereffect damage. (s-x0.1~.le.2x.2s) W h a t is the explanation for this residual and considerable heterogeneity in the stored, but not immediately-planted, seeds? It must be some factors which are operating after, but only slightly or not at all during irradiation. T h e total amount of this damage developing during storage is very large: it is usually about five times but m a y be as much as ten or more times as great as the 'immediate' damage; that is, the 50 per cent height doses for stored seeds m a y be ~ - ~ or less that for seeds handled identically but planted immediately.(S.12.13. TM Hence, in stored seeds, most of the damage detected ( ] - ~ or more) is developed after irradiation. It is known that long-lived free radicals are present in these dry seeds postirradiation, and that damage develops proportional to the size and decay of this ESRdetected radical population; it is also clear that the free radicals present post irradiation engage in a long series of radical-molecule reactions which m a y lead to development of the storage damage (see references 7-10, 16, 18, 24, 25, and earlier papers). I f for convenience we call the immediate damage 'direct' and the stored damage 'indirect', then it seems reasonable that the

I R R A D I A T E D BARLEY SEEDS latter should be m u c h m o r e susceptible to modification b y i n t r a c e l l u l a r conditions t h a n the former. T o categorize the two, the d i r e c t i m m e d i a t e d a m a g e arises from very shorttime, short d i f f u s i o n - d i s t a n c e interactions of small molecule or r a d i c a l p r o d u c t s ; while the indirect stored d a m a g e comes from long-time, long d i f f u s i o n - d i s t a n c e interactions of large a n d less reactive molecule or r a d i c a l products. Factors which could a p p r e c i a t e l y m o d i f y the reactions in the l a t t e r case could be w i t h o u t effect or m i n i m a l in the former. F o r e x a m p l e , storage d a m a g e can be c o m p l e t e l y s t o p p e d b y low ( - - 7 6 ° C or lower) t e m p e r a t u r e s or slowed by d r y n e s s - - f a c t o r s which increase r i g i d i t y of the cellular m a t r i x - - b u t these modifiers have far less effect on a m o u n t o f i m m e d i a t e d a m a g e . Some of these factors which increase, decrease, or t e r m i n a t e storage d a m a g e , a n d which do not a p p l y or are insignificant to i m m e d i a t e d a m a g e , could be heterogeneous w i t h i n the seeds a n d responsible for the d e t e c t e d heterogeneity. W e are u n a b l e to say w h a t the p e r t i n e n t factors are. W e can only state they are not the m o r e obvious ones of h e t e r o g e n e i t y in dose, in the seeds themselves, or in seed oxygen content, p r o b a b l y not in m o i s t u r e c o n t e n t o f the seed as a whole, a n d not from u n r e l i a b i l i t y of the seedling-height criterion itself.

13

3. CALDEGOTT, R. S. (1954) Inverse relationship between the water content of seeds and their sensitivity to X-rays. Science. 120, 809-810. 4. CALDECOTT R. S. (1955) Effects of hydration of X-ray sensitivity in Hordeum. Radiation Res. 3, 316-330. 5. CALDECOTTR. S. (1961) Seedling height, oxygen availability, storage and temperature: Their relation to radiation-induced genetic and seedling injury in barley. In Effects of Ionizing Radiations on Seeds pp. 3-24. IAEA, Vienna. 6. CALDECOTT R. S., FROLIK E. F. and MORRIS 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, 804-809. 7. CONGER A. D. and RANDOLPH Ik~i. L. (1959) Magnetic centers (free radicals) produced in cereal embryos by ionizing radiation. Radiation Res. 11, 54-66. 8. CONGERA. D. (1961) Biological after-effect and long-lived free radicals in irradiated seeds. J. Cellular Comp. Plo,siol. 58, Suppl. 1, 27-32. 9. CONGER A. D. (1963) Chromosome abe,-rations and fi'ee radicals. In Radiation-lnduced Chromosome Aberrations. (Edited by WOLFF S.) pp. 167-202. Columbia University Press, N.Y. 10. CONGER A. D. (1966) Biological damage and free radicals in irradiated seeds. In Symposium Electron Spin Resonance and the Effects of Radiation on Biological Systems (Edited by StaPES W.) pp. 177189. NAS-NRC Nucl. Sci. Ser. Rep't. 43. 11. CONGER B. V., NXLANR. A., KONZAK C. F. and METTER S. (1966) The influence of seed water Acknowledgements--Most of the experimental work content on the oxygen effect in irradiated barley was done while the authors were members of the seeds. Radiation Botany 6, 129-144. Nuclear Sciences Group at the University of Florida, 12. CURTISH.J., DELIHASN., CALDECOTTR. S. and Gainesville, Florida. A part of this was the graduate KONZAKC. F. (]958) Modificationsof radiation work of the junior author supported at that time by a damage in dormant seeds by storage, Radiation Graduate School and by a Nuclear Sciences FellowRes. 8, 526-534. ship of the University of Florida; the remainder is 13. EHRENBERG L. (1955) The influence of postto be published elsewhere. irradiation factors on effectsproduccd in barley Radiobiol. Syrup. Proc. pp. 285-289. Liege, 1954. 14. EHRENBERG L. (1955) The radiation-induced growth inhibition in seedlings. Botan. Notiser. 108, 184-215. REFERENCES 15. EHRENBERGL. and NYBOM N. (1954) Ion density I. ADAMSV. D., NILAN R. A. and GUNTHARDT and biological effectiveness of radiations. Acta H. M. (1955) After-effectsof ionizing radiation Agr. Scand. 4, 396-418. in barley--I. Modificationby storage of X-rayed 16. EHRENBERG A. and EHRENBERG L. (1958) The seeds. A preliminary report. Northwest Sci. 29, decay of X-ray induced free radicals in plant I01-I08. seeds and starch. Arkiv Fysik. 14, 133-141. 2. BOZZlNIA., CALDECOTTR. S. and NORTHD. T. 17. KONZAK C. F., NIL~d~ R. A., HARLE J. R. and (]962) The relation of seedling height to genetic HmNER R. E. (1961) Control of factors affecting injury in X-irradiated barley seeds. Radiation the response of plants to mutagens. Brookhaven Res. 16, 764-772. Symp. Biol. 14, 128-157.

14

ALAN D. CONGER and HARLAN Q. STEVENSON

18. LOFROTIfG., EHRENBERGA. and EHRENBERG L. (1964) Analysis of radiation-induced electron spin resonance spectra in plant seeds. Radiation Botany 4, 455-467. 19. NATARAJAN A. T. and AHNSTR6M G. (1961) Oxygen saturation and dry seed irradiation. aVaturwissenschaften 48, 698-699. 20. NILAN R. S. (1954) Relation of carbon dioxide, oxygen and low temperature to the injury and cytogenetic effects of X-rays in barley. Genetics 39, 943-954. 21. NXLANR. S., KONZAKC. F., LEGAULTR. R. and HARLE J. R. (1961) The oxygen effect in barley seeds. In Effects of Ionizing Radiations on Seeds pp. 139-154. IAEA, Vienna. 22. NILAN R. S., KONZAK C. F., HARLE J. R. and HEINER R. E. (1962) Interrelation of oxygen, water and temperature in the production of radiation-induced genetic effects in plants. In Strahlewirkblg und Milieu. Strahlentherapie Suppl. 51, 171-181.

23. OSBORNET. S., LUNDENA. O. and CONSTANTIN M. J. (1963) Radiosensitivity of seeds--III. Effects of pre-irradiation humidity and y-ray dose on seeds from five Botanical families. Radiation Botany 3, 19-28. 24. POWERSE. L. (1961) Reversibility of X-irradiation-induced effects in dry biological systems. 07. Cellular Comp. Physiol. 58, Suppl. 1.13-25. 25. RANDOLPHM. L., I-IEDDLEJ. A. and HozzuJ. L. (1968) Dependence of ESR signals in seeds on moisture content. Radiation Botany 8, 339-343. 26. TALLANTmE A. and POWERS E. L. (1963) Modification of sensitivity to X-irradiation by water in Bacillus megaterium. Radiation Res. 20, 270-287. 27. WOLFFS. and LUIPPOLDH. E. (1956) Obtaining large numbers of metaphases in barley roots. Stain Technol. 31, 201-205. 28. WOLF S., and SICARD A. M. (1962) Post irradiation storage and the growth of barley seedlings. In Effects of Ionizing Ra~ations on Seeds pp. 171-179 IAEA, Vienna.