Influence of oxygen at high pressure on the induction of damage in barley seeds by gamma radiation

EnviromnemalandExpertraentMBola~.,Vol. 20, pp. 11 to 19

0098-8472180/0101-0011 $02.00/0

© Pergamon Press Ltd. 1980. Printed in Great Britain

I N F L U E N C E OF O X Y G E N AT H I G H P R E S S U R E ON T H E I N D U C T I O N OF D A M A G E IN BARLEY SEEDS BY G A M M A R A D I A T I O N * E,. D O N A L I ~ O N ' ] , I~ A. NILAN and C:. F. KONZAK Department of Agronomy and Soils and Program in Genetics, Washington State University, Pullman, Washington 99164, U.S.A. (Received 6 November 1978; accepted in revisedform 26 Mar 1979)

DONALDSON E., NIt~N R. A. and KONZAK C. F. Influence of oxygen at high pressure on the induction of damage in barley seeds by gamma radiation. ENVIRONMENTAL AND EXPERIMENTAL BOTANY20, 11-19, 1980.--The influence of high pressure oxygen (HPO) before, during and after irradiation on seedling injury (percent reduction in seedling height relative to the nonirradiated controls) was investigated using Himalaya (C.I. 620) barley seeds. Seeds were adjusted to water contents of 2-14% by storage in vacuum desiccators over calcium oxide or mixtures of glycerol and water and then irradiated in vacuo or under various oxygen tensions with 6o(30 gamma rays. After irradiation, the seeds were soaked at approximately 0°C in oxygen or nitrogen bubbled water. Treatment effects were recorded as M a seedling injury. Seeds exposed to HPO before, during or after irradiation followed by soaking in oxygen or nitrogen expressed two or three times more damage than irradiation in vacuo followed by soaking in oxygenated water. That a reaction between oxygen and radiation-induced sites probably occurs before the seeds are soaked was demonstrated by the failure to remove completely the effect of HPO by vacuum between HPO treatment and irradiation. The results indicate that placing the seeds under HPO may increase the rate and extent of reactions which occur during post-radiation storage of seeds in the presence of oxygen. The increase in damage associated with oxygen soaking (oxygen-dependent damage) is partially lost during aerobic storage and is largely pre-empted when seeds are placed under HPO. This decrease in oxygen-dependent damage is accompanied by an increase in damage occurring with nitrogen soaking, suggesting that the reaction which leads to damage was initiated before soaking and to the same oxygen sensitive sites. INTRODUCTION THE INFLUENCE o f increased pressures of oxygen ( H P O ) on post-radiation d a m a g e in ' d r y ' biological systems is not well understood. Earlier investigations have shown that irradiation in H P O will enhance radiobiological d a m a g e above that found when seeds i r r a d i a t e d in vacuo are soaked in oxygenated w a t e r (oxygen

soaking). BERG(2) o b t a i n e d no further d a m a g e from oxygen soaking following i r r a d i a t i o n in H P O . H P O must increase the a m o u n t of oxygen in the seeds, which should increase the efficiency of the o x y g e n - o x y g e n sensitive site ( O S S ) reaction. However, the influence of H P O on the rate o f o x y g e n - O S S reaction responsible for the oxygen e n h a n c e m e n t of d a m a g e in

*Scientific Paper No. 5061. College of Agriculture Research Center, Washington State University, Pullman. Research supported in part by a grant from the U.S. Energy Research and Development Administration, Contract EY-76-S-06-2221, and conducted under Projects 1068 and 1568. ERDA RLO-2221-T2-37. tPresent address: Dry Land Research Unit, Lind, Washington 99341, U.S.A. 11

12

E. DONALDSON, R. A. NILAN and C. F. KONZAK

irradiated seeds has not been determined. Furthermore, the radiobiological results indicating an oxygen-dependent reaction before soaking ~2'21~ lack necessary controls, although electron paramagnetic resonance (EPR) analyses indicate that the reaction probably

bubbled during soaking. Soaking at 0cC retards metabolic processes, provides the m a x i m u m concentration of dissolved oxygen, t13~ and extends the development time for oxygendependent d a m a g e J 7)

Occurs.(.,'], 1 1,12,19,26)

Irradiation under pressure

This study was initiated to determine when and to what extent H P O (136arm) affects the magnitude of the oxygen-dependent portion of damage occurring in irradiated seeds.

Seeds of the appropriate water content were removed from vacuum desiccator storage and placed in perforated polyethylene vials, then into pressure chambers ~2) under a plastic hood flushed with the gas to be used for pressure. T h e chambers were continually flushed with the pressure gas until they were sealed. For pressure treatments only, the water content of the seeds in the desiccator was assumed to be the water content of the seeds during treatment. During a 30-day pressure treatment of seeds containing 3.6°o water, a moisture increase of only 0.303 was obtained. BERo~2) also found that the water content of the seeds did not change appreciably during a pressure treatment in the chambers. All exlaeriments with seeds irradiated under pressure also included treatments of seeds in glass vials in a partial vacuum tbr comparison. These were placed in a pressure chamber prior to irradiation. Thus, all seeds in an experiment were exposed to similar doses of irradiation regardless of the pressure condition.

MA'IT.,B.IALS A N D G E N E R A L M E T H O D S

Preparation and treatment of biological material Medium-sized (passed through 2.78 x 19.05mm and ~zollected in 2 . 3 8 x 19.05mm screen) seeds (caryopses) of the hulless barley (Hordeum vulgare) cultivar ' H i m a l a y a ' (C.I. 620) were used in this study. Seed water contents between 2 and 8°.o were obtained by storage over calcium oxide in a v a c u u m desiccator. Seeds with 1 0 - t 4 % water content were obtained by storage over glycerin-water mixtures in a v a c u u m desiccator. ~7'16~ Since active pumping (evacuation) removes water from more moist seeds, the final water content of all seeds was determined after pumping. ~22)

Irradiation under partial vacuum Seeds were placed in 1 0 m m diameter glass vials, attached to a glass manifold vacuum system ~ls'x6'22) and actively pumped to create as high a vacuum as possible. T h e duration of pumping partially depended on the initial water content of the seeds. The 10-140:o water content seeds were actively pumped for at least 0.5hr and then allowed to equilibrate on the system for at least 10hr before removal. Seeds containing 6-80~ water were actively pumped for at least 4 hr, and those containing 2-4°o water for at least 10hr. After the evacuation period, the vials were sealed under vacuum. The seeds were irradiated in the sealed glass vials. After irradiation, the evacuated glass vials were opened and the seeds placed in perforated polyethylene vials, which were then placed into 0 ° + 0 . 5 ° C oxygen or nitrogen bubbled distilled water. The water was bubbled for at least 30 min prior to seed soaking, and continuously

Irradiation source and procedure Irradiation was conducted in the Washington State University 6°Co g a m m a facility. ~4'1°~ A new 6°Co loading was added during the course of the study (previously 2.1 krad/min, now 15). In experiments without pressure treatments and when only dry seeds (6" 0 moisture or less) were used, they were irradiated at room temperature and taken from the facilitv to the laboratory tbr soaking. The handling of seeds in experiments containing more moist seeds is covered under specific methods.

Measurement of biological damage Seedlings from soaked seeds were cultured as previously describedJ 1~'2°) After culturing for 5-6 days, the length of the first seedling leaf was measured to the nearest millimeter. Unless

INFLUENCE OF OXYGEN .VI I I l ( ; l l I'RI.;SSURE otherwise stated, three i n d e p e n d e n t replications of 50 seeds each were used per treatment. M e a n seedling height for each t r e a t m e n t replication, coefficient of variation and percent seedling injury (percentage reduction in M1 seedling growth relative to the control) were calculated with the Seedling H e i g h t Calculating P r o g r a m for the I B M 360 C o m p u t e r of the W a s h i n g t o n State University C o m p u t i n g Center. Analysis of variance for seedling height of factorial experiments was c o m p u t e d with the B M D O 8 V P r o g r a m ( t ) from the t r e a t m e n t replication means.

RESULTS AND SPECIFIC METHODS Movement o f oxygen into seeds

Several experiments conducted d u r i n g the course of this study indicated the rate at which oxygen enters to react in i r r a d i a t e d seeds. O n l y one e x p e r i m e n t was c o n d u c t e d specifically for this purpose. Seeds of 3.8°0 water content were i r r a d i a t e d with 20, 40 and 6 0 k r a d in a partial v a c u u m in glass vials. T h e vials were opened and the seeds exposed to air at room t e m p e r a ture for up to 120rain before the start of soaking. T h e control sample for each dose was transferred to the soaking w a t e r u n d e r a nitrogen flushed hood. (5'v'16) All samples were soaked in n i t r o g e n - b u b b l e d water. T h e results (Table 1) indicate that u n d e r the conditions in the l a b o r a t o r y , insufficient oxygen entered or reacted in the seeds in tess than 15 rain to cause a significant a m o u n t of additional injury.

13

W h e n seed water content was raised to 9.80 o in a separate experiment, 5 min exposure to air after i r r a d i a t i o n in vacuo did not significantly increase the injury. T h e seeds in this experiment were frozen to - 7 8 ° C i m m e d i a t e l y after irradiation and allowed to w a r m d u r i n g exposure to air before nitrogen soakingJ 5) T h e seeds were frozen to prevent decay of OSS d u r i n g transport to the l a b o r a t o r y as occurs in vacuo in more moist seeds. A n increase in d u r a t i o n of exposure to and partial pressure of oxygen before soaking significantly increased the d a m a g e with nitrogen soaking, as found in an experiment where 2.30o water content seeds were i r r a d i a t e d in a partial v a c u u m with 5 k r a d . T h e seed samples were exposed to oxygen in the pressure chambers for 1 and 2 days at atmospheric pressure, and for 15rain and 2 h r at both 34 and 136arm. Seedling injury was calculated from two indep e n d e n t replications of 150 seeds for each treatment. W h e r e the i r r a d i a t e d seeds were exposed to oxygen at atmospheric pressure for one day, seedling i n j u w following nitrogen soaking was increased by 33.8°o c o m p a r e d to i r r a d i a t e d seeds not exposed to oxygen. This was accompanied by a decrease in the soaking oxygend e p e n d e n t d a m a g e by 45.3°,'o c o m p a r e d to the irradiated seeds not exposed to oxygen prior to soaking. A n additional d a y of exposure to oxygen did not increase the a m o u n t of d a m a g e obtained with either oxygen or nitrogen soaking. W h e n seeds were exposed to oxygen at

Table 1. Time required for o.(vgen to enter barley' seeds of 3.8'% water content, as shown by percent seedling injur~,, when exposed to air after irradiation in partial aiJ t(,,,bt vacuum

Duration of exposure to air (rain) < 0.5 Dose (krad)

7.5

Control+ (cm)

• 0 20 40 60

14.7 14.4 11.1 6.5

15

30

60

120

--0.20 17.77" 11.68 15.34"

0.34 16.79" 5.75 5.98

Seedling injury (",,) -2.58 0.49 1.62 -- 1.84

0.75 1.95 --4.85 1.69

0.0 7.25 4.49 --2.45

--1.83 12.13" 1.98 3.68

i

*Significantly different from the controls at the 0.05 level. tSeedling heigh~ of treatments transferred to the soaking water under the nitrogen flushed hood.

14

E. DONALDSON, R. A. NILAN and C. F. KONZAK

34atm for 15min, the injury after nitrogen soaking was increased to 74% and the accompanying reduction in soaking oxygen-dependent damage was 90.6%. Neither higher pressure nor longer exposure significantly increased the damage. Removal of oxygen from seeds by vacuum pumping To demonstrate that oxygen-dependent damage (ODD) could occur before soaking, seeds of 2.3% water content were held under 136atm oxygen pressure for 8.25 hr and irradiated with 7.5krad (Table 2). Some seeds were vacuum pumped between pressure and radiation treatments and other pressure-treated seeds were vacuum pumped after radiation treatment. Other seeds were irradiated in a partial vacuum and further vacuum pumped after irradiation. Non-pressure treatments were vacuum pumped for 2 days before irradiation and 8 days after irradiation. HPO-treated seeds were vacuum pumped for 8 days. Samples pumped after irradiation were irradiated 8 days before samples which were not, so that all samples could be soaked at the same time. One sample from each treatment combination was soaked in oxygen and another in nitrogen-bubbled water. Two replications of 150 seeds each per treatment were used.

The results (Table 2) indicate that, if oxygen pressure were not involved, pumping following irradiation tended to allow more oxygenindependent damage (1.8 compared to 11.0% injury) to occur but did not reduce the amount of oxygen-dependent damage. When a pressuretreated sample was pumped before irradiation (P-V-R), the soaking oxygen-dependent damage (ODD) partially returned (37.2% ODD), but pumping after irradiation ( P - R - V ) had practically no influence (0.0% ODD). At an injury level of 75%, only slight additional injury could be expected from oxygen soaking. However, if the factors causing damage from irradiation under oxygen pressure could have been removed, the injury level would have been lower. This experiment indicates that the soaking ODD could be obtained if oxygen were removed by vacuum pumping between the oxygen pressure treatment and the irradiation treatment, but not by vacuum pumping after irradiation. Influence of exposure to HPO on the soaking ODD Seeds of 2.2, 5.1, 10.1 and 14% water content received exposures of 0, 5, 10 or 15 krad in a partial vacuum and under H P O at 136 atm to estimate the interaction between seed water content and radiation exposure on the soaking

Table 2. Seedling height of the controls (era) and percent seedling injury for oxygen pressure-irradiation combinations on barley seeds of 2.3% water content i

Soaking atmosphere Order of treatment conditions* V P P- V V-R V-R-V P-V-R P-R P-R- V

02

N2

12.6 cm 9.5 cm 9.8 cm

12.4 cm 9.7 cm 9.0 cm 48.7 54.7 58.6 71.8 76.4

Soaking oxygen dt'pt'ndc,II damage (",,)t

! .8 11.0 29.7 63.4 75.5

45.6 43.2 37.2 9.4 0.0

*Where P is 136 atm oxygen pressure for 8.25 hr, released before irradiation; V is vacuum pumped; and R is 7.5 krad. t Tn-- To where T and C are seedling height of treatment and control, respectively, and o and n x 100, are oxygen and nitrogen soaking, respectively. (Cn + Co)/2

INFLUENCE OF OXYGEN AT HIGH PRESSURE ODD and the pressure ODD. The duration of pressure treatments was 16hr before and 8hr after irradiation. Seeds irradiated in a partial vacuum were soaked immediately after irradiation to maximize the possible soaking ODD. A balanced incomplete block design was used. No significant effect was obtained for the blocks so they were combined in the analysis. More ODD resulted from irradiation of seeds under H P O than from irradiation of seeds in a partial vacuum and then soaked in oxygenbubbled water (Table 3). Virtually no additional oxygen-dependent damage could be obtained when pressure irradiated seeds were soaked in oxygen-bubbled water. Generally, oxygen-independent damage (OID) increased with an increase in radiation exposure and decreased as seed water content increased. A second experiment was conducted using seeds of 2.3% water content. The seeds were put under 136atm oxygen pressure for 16hr, irradiated, left under pressure for 8 h r more, and soaked overnight in oxygen- or nitrogenbubbled water. The results (Fig. 1) show that the soaking oxygen-dependent damage was removed (within experimental error) by the pressure ODD. Exposures of 0.5, 1, 2, 3 and 5 krad were given.

lnfluence of oxygen concentration on the pressure oxygen-dependent damage The object of this experiment was to study the concentration of oxygen needed for the pressure O D D during irradiation. Seeds containing 2.0°,,o water were placed in the chambers under a plastic hood flushed with oxygen. The chambers were removed from the hood and filled to a predetermined pressure for each cylinder with nitrogen in the usual way. The lines were then flushed and the chambers brought to 136atm with oxygen. The intention was to obtain gas mixtures of about 1.0, 6.25, 25, 50 and 100% oxygen. The seeds were pressure treated for 16hr before and 8hr after receiving 4krad. The experiment was then conducted in the usual way with only one replication per treatment. The minimum oxygen concentration obtainable by the system used was calculated to be

15

23% seed water O x y g e n soaking

8O

60 5C

E

30

g 2o "~ ~0 0 CE

I 005

I I

I 2

I 3

I 5

Radiation exposure, krod

Fro. l. Percent reduction in seedling height compared to non-irradiated pressure treated barley seeds of 2.3°.o seed water content subjected to 136atm high oxygen pressure (for 24hr) and several exposures ol radiation. about 1°o. All samples yielded about the same amount of injury (Table 4). The samples soaked in nitrogen-bubbled water showed less injury than those soaked in oxygen-bubbled water, indicating a slight soaking ODD. DISCUSSION AND CONCLUSION Increasing the oxygen pressure in barley seeds during irradiation could be expected to increase reaction efficiency between oxygen and oxygen sensitive sites (OSS). NILAN et al. (23~ .added oxygen to dry seeds and removed it by vacuum pumping. Results obtained after subsequent nitrogen soaking led them to conclude that the oxygen had to be dissolved in water to react with radiation products. BERG,(z~ after obtaining no increase in damage from oxygen soaking of seeds which had been irradiated under HPO, concluded that the availability of oxygen during irradiation resulted in the formation of damaging products prior to soaking. Another interpretation of Berg's results is possible. Seeds could absorb all the oxygen needed for soaking ODD when subjected to oxygen pressure before soaking. (7~ In the present study, when seeds were placed under H P O and the pressure released before irradiation, the results were the same as from experiments where seeds were irradiated while under oxygen pressure. However, the observation that BERG(2~ was un-

Table 3. Oxygen dependent and independent damage 1'!o injury} resultingfrom irradiation of barley seeds in vacuum and under oxygen pressure Without pressure

With pressure

Seed water content {"o)

Irradiation exposure (krad)

2.2

5 l0 15

40.9 48.0 62.2

14.3 25.9 17.0

--3.1 -9.4 0.0

67.0 49.5 67.0

5.1

5 10 15

7.4 39.3 63.1

- 1.6 0.0 1.6

4.3 -0.9 2.6

73.9 85.2 90.4

5 |0 15

1.5 15.6 32.0

3.0 2.3 9.1

-6.7 - 1.9 1.9

13.2 55.7 68.9

5 10 15

- 1.5 -0.7 0.0

-2.3 2.3 1.5

-- 14.3 5.9 2.5

-0.9 6.8 14.5

10.1

14.0

O 2 Dep D a m

O2 Ind Dam

O2 Soak Dep Dana

O 2 Pres Dep Dam

© Z >.

©

:Z

> ;<

Cn-Tn

0 2 Ind Dana--Oxygen independent danmge. - - x

100.

£n

0 2 l)ep Dam - O x y g e n dependent damage,

if3

To - 'In ICo+Cn)/2

x 100.

(12 Soak l)ep l ) a m - - ( ) x y g e n dependent damage remitting from soaking in oxygen saturated water following irradiation under oxygen

0

pressure.

N

>

PrO - PaN

x 1(X).

~PCo - P t n ) / 2

0 2 Pres l)ep l)am --Oxygen pressure dependent danmge resuhing from irradiating under oxygen pressure.

( P C n - P T n ) - ( C n - Tn) PCn

x 100,

Where C is the non-irradiated control and T is the irradiated treatment seedling height, respectively; n and 0 denote nitrogen and oxygen soaking, respectiw~|y, and P in ti'ont of a symho[ indicates that the sample was treated with oxygen pressure. Data are averages of three replications.

INFLUENCE OF OXYGEN AT HIGH PRESSURE

17

Table 4. Influence of ox~,gen concentrations ~"o ) on the pressure ODD during irradiation of barler seed Reduction in seedling height (°'o)* Oxygen concentration ("o) O Pres 1.0 6.25 12.5 25.0 50.0 100.0

Soak

Atm

02

N2

O 2 Soakt Dep Dam

37.2 87.3 92.7 92.0 85.3 89.1 91.5

5.7 83.8 89.1 83.1 85.7 83.2 85.6

33.5 3.5 3.6 8.9 - 0.4 5.9 5.9

O 2 Pres+ Dep Dam

Total oxygen dependent damage [,"o)

-78.1 83.4 77.4 80.0 77.5 79.9

32.5 81.6 87.0 86.3 79.6 83.4 85.8

*Based on one replication of 50 seeds per treatment. Seeds of 2.0°0 water were irradiated with 4krad while under 136 atm pressure, 8 hr before release of the pressure. ?Percent reduction in seedling heights of oxygen soaked seeds minus nitrogen soaked seeds. .+Percent reduction in seedling heights of pressure treated seeds minus non-pressure treated seeds both soaked in nitrogen. able to reduce the d a m a g e by v a c u u m p u m p i n g d a m a g e decreased as w a t e r content increased to 14°o, where at least 15krad were required to the seeds between irradiation and soaking would tend to support his conclusion. obtain a significant increase in pressure O D D NATARAJANa n d AHNSa'R6~~21) also found oxy- However, the soaking O D D is not significant at gen effects difficult to remove with v a c u u m a n y r a d i a t i o n exposure with seeds this high in p u m p i n g after saturation with pressure and water content, except u n d e r special conc a m e to the same conclusion as BER6. t2) T h e y ditions) xs'25) Generally, the pressure O D D was did not explicitly state whether they tried to 2-3 times greater than the non-pressure O D D remove the oxygen before or after irradiation. (Table 3). I n studying the O D D by bacterial T h e conclusion that 'oxygen' could not be spore survival while irradiated d~- or in water, POWERS,(x4) and removed is further supported by the change or EWING, (13) EWING and elimination of the E P R signal with exposure of TALLEr~TINX and POWERS~2v) separated the dai r r a d i a t e d m a t e r i a l to oxygenJ 6' 11.19.26) m a g e into three classes. Class I was oxygen T h e effects of H P O on seeds can be modified i n d e p e n d e n t , Class I I occurred when oxygen by v a c u u m p u m p i n g before, but not after irra- was present d u r i n g irradiation, and Class I I I diation (Table 2). This is the best biological occurred when oxygen was present after evidence for the occurrence of an oxygen--OSS irradiation. I n the present investigation with barley seeds, reaction before soaking. However, some water is present even in very d ~ seeds and m a y be i r r a d i a t i o n of seeds saturated with oxygen by reflected bv the a m o u n t of o x y g e n - O S S re- pressure (Class I I ) virtually eliminated the action. Regardless of the reaction, irreversible soaking O D D (Class I I I ) u n d e r most conditions steps seem to occur before soaking. studied TM (Table 2), as m a y be the case with A t the same r a d i a t i o n exposure, seeds u n d e r spores if the same spores were studied for both HPO exhibit considerably more oxygen- classes of d a m a g e . This p r e - e m p t i o n of the d e p e n d e n t d a m a g e than those in a vacuunq soaking O D D seems to be i n d e p e n d e n t of seed followed by oxygen soaking/2) T h e increase in water content and r a d i a t i o n exposure (Table 3, d a m a g e occurring in H P O - t r e a t e d samples was Fig. 1). This does not mean that the soaking influenced by w a w r content similar to the soak- and pressure O D D reactions are necessarily the ing O D D (q',b~l, 3). T h e o x y g e n - d e p e n d e n t same, ~14) b u t only that a reaction occurring

18

E. DONALDSON, R. A. NILAN and C. F. KONZAK

d u r i n g i r r a d i a t i o n makes the OSS unavailable for reaction during soaking, i.e. the. reactants m a y be the same but the subsequent reactions m a y be quite different. D u r i n g irradiation, gaseous oxygen is p r o b a b l y the reactant, while d u r i n g soaking, the reactant is dissolved oxygen and m a y diffuse into the cells only as carried by the water. Since the d a m a g e is much greater with the H P O , the oxygen concentration with high pressure might be greater in seed embryos and m a y react i m m e d i a t e l y with more sites or more types of sites than dissolved oxygen. Some OSS m a y be lost d u r i n g soaking before oxygen has a chance to react with them. T h e sites available for an oxygen reaction d u r i n g irradiation a p p e a r to remain available in dry seeds for some time after irradiation, iT's) This allows the change in O D D with time to be used to study the entry rate of oxygen into seeds. W i t h oxygen pressure alone, the increase in d a m a g e a p p e a r e d to level off after about 8 hr of exposure, much as observed by NATARAJAN and AHNSTR6M.t2t) I r r a d i a t e d seeds containing 2.3% water left exposed to oxygen at atmospheric pressure for one d a y showed a 45.3% decrease in the soaking O D D and an increase of 33.8% in observed d a m a g e after nitrogen soaking. No further change resulted from additional exposure time. W i t h higher oxygen pressures, the reactions of O S S with oxygen were accelerated and more complete. Very d r y i r r a d i a t e d seeds stored in aerobic conditions followed by nitrogen soaking show increased d a m a g e with storage time, Ct's'2*~ while very dry i r r a d i a t e d seeds stored in a v a c u u m do not. C7) A b o u t 15 min are required for any effect to be observed when seeds were stored in air under laboratory conditions (Table 1). T h e effect of increased oxygen pressure ( o x y g e n c o n c e n t r a t i o n ) would be to increase the rate and extent of oxygen reactions which would occur d u r i n g aerobic storage of irradiated seeds. S o a k i n g O D D is partially removed during storage and almost completely removed by high oxygen pressure. T h e decrease in the soaking O D D is a c c o m p a n i e d by an increase in d a m a g e with nitrogen soaking, suggesting that the d a m a g e was done before soaking and to the same OSS.

REFERENCES 1. ADAMSJ. D. and NILAN R. A. (1958) Aftereffects of ionizing radiation in barley. II. Modification by storage of X-irradiated seeds in different concentrations of oxygen. Radiat. Res. 8, 111-122. 2. BERO C. C., NILAN R. A. and KONZAK C. F. (1965) The effect of pressure and seed water content on the mutagenic action of oxygen in barley seeds. Murat. Res. 2, 263-273. 3. BHASKARAN S. and K6HNLEIN W. (1964) ESR studies on plant seeds of differential radiosensitivity. II. Effect of oxygen and nitric oxide at different temperatures. Radiat.. Bot. 4, 291-298. 4. BOTCHEK C. M., KONZAKC. F. and NILAN R. A. (1964) Design of the 6°Co facility at Washington State University. I E E E Trans., PEP 8~ 2-8. 5. BOTTINO P. J. (1969) The effect of radiation energy on the oxygen effect in irradiated barley seeds. Ph.D. Thesis, Washington State University. 76 p. 6. CONOER A. D. (1966) Biological damage and free radical in irradiated seeds. Pages 177-189 in Electron spin resonance and the effects of radiation on biological systems. Natl. Acad. Sci.-Natl. Res.

Council, Publ. 1355, Washington D.C. 7. CONOER B. V., NmAN R. A., KONZAK C. F. and METTER S. (1966) The influence of seed water content on the oxygen effect in irradiated barley seeds. Radiat. Bot. 6~ 129-144. 8. CURTIS H. J., DELIHAS N., CALDECOTT R. S. and KONZAK C. F. (1958) Modification of radiation damage in dormant seeds by storage. Radiat. Res. 8~ 526-534. 9. Dixon W. J. (1964) Analysis of variance, BMDO8V. In Biomedical computer programs. Health Services Computing Facility, University of California, Los Angeles. 10. DONALDSON E. (1970) The interaction of some factors influencing the oxygen effect in irradiated barley seeds. Ph.D. Thesis, Washington State University. 114 p. 11. EHRENBERG A. (1961) Research on free radicals in enzyme chemistry and radiation biology. Pages 337-350 in M. S. BLots, JR., ed. Free radicals in biological systems. Academic Press, New York. 12. EHRET C. F., SMALLER B., POWERS E. L. and WEBB R. B. (1960) Thermal annealment and nitric oxide effects on free radicals in Xirradiated cells. Science 132, 1768-1769. 13. EWING D. (1978) Additive sensitization of bacterial spores by oxygen and p-nitroacetophenone. Radiat. Res. 73, 121-136.

INFLUENCE OF OXYGEN AT H I G H PRESSURE

14. EWING D. and PowERs E. L. (1976) Irradiation of bacterial spores in water: three classes of oxygen-dependent damage. Science l~t, 10491051. 15. HARLE J. R. (1963) Modification of radiobiological oxygen effects in very dry barley seeds. Ph.D. Thesis, Washington State University. 68 p. 16. KONZAK C. F., BOTTINO P. J., NII~N R. A. and CONOER B. V. (1968) Irradiation of seeds: A review of procedures employed at Washington State University. Pages 83-96 in Neutron irradiation of seeds II. International Atomic Energy Agency, Vienna. 17. KONZAK C. F., MIKAELSEN K., SIOURBJfRNSSON B. and BURa'SCHZR A. (1967) Recommended standard procedures for irradiating, cultivating and measuring cereal seeds to determine the effects of neutron irradiation in the neutron-seedirradiation programme. Pages 103-107 in Neutron irradiation of seeds. Technical Reports Series No. 76, International Atomic Energy Agency, Vienna. 18. KONZAKC. F., NILAN R. A., LEGAULTR. R. and HmNER R. E. (1961) Modification of induced genetic damage in seeds. Pages 155-169 in Effects of ionizing radiations on seeds. International Atomic Energy Agency, Vienna. 19. LION M. B., KIRBY-SMITH J. S. and RANDOLF M. L. (1961) Electron spin resonance signals from oxygen-exposed lyophilized bacteria cells. Nature (London) 192, 34-36. 20. MYmLL R. R. and KONZAK C. F. (1967) A new technique for culturing and measuring barley seedlings. Crop Sci. 7, 275-276.

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NATARAJANA. T. and AHNSTROM G. (1961) Oxygen saturation and dry seed irradiation. aVaturwissenshaften 48, 698-699. 22. NILAN R. A., KONZAK C. F., HAglX J. R. and HEINER R. E. (1962) Interrelation of oxygen, water and temperature in the production of radiation induced genetic effects in plants. Pages 171-182 in Strahlenwirkung mad milieu (Suppl. to Strahlentherapie, Vol. 51 ). Urban & Schwarzenberg, Munich. 23. NXLAN R. A., KONZ^K C. F., HARI~ J. R. and LEGAULT R. R. (1961) The magnitude of the oxygen effect in barley seeds. Pages 139--154 in Effects of ionizing radiations on seeds. International Atomic Energy Agency, Vienna. 24. SIRE M. W. and NIta~N R. A. (1959) The relation of oxygen post treatment and heterochromatin to X-ray-induced chromosome aberration frequencies in Crepis capillaris. Genetics 44, 21.

124-136.

25. SPEr~CE R. K. (1965) The influence of sodium azide on the biological effects of ionizing radiation in moist barley seeds. M. S. Thesis, Washington State University. 59 p. 26. SWARTZ H. M. and RaCHAgVSONE. C. (1967) A correlation between radiation-induced free radicads and survival in micro-organisms exposed to •-mercaptoethylamine under oxygen or nitrogen. 1,u. y. Radiat. Biol. 12, 75--88. 27. TALta~rcram~ A. and Powr.RS E. L. (1963) Modification of sensitivity to X-irradiation by water in Bacillus megatherium. Radiat. Res. 20, 270287.