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The interaction of oxygen, radiation exposure and seed water content on γ-irradiated barley seeds
Environmental and Experimental Botany, Vol. 19, pp. 153 to 164.
0098-8472/79/0801-0153 $02.00/0
c; Pert~amon Press Ltd. 1979. Printed in Great Britain
THE INTERACTION OF OXYGEN, RADIATION EXPOSURE AND SEED WATER CONTENT ON 7-IRRADIATED BARLEY SEEDS* E. DONAI.I~SON, R. 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 13 June 1978; in revisedform 9 November 1978; accepted 13 November 1978) DONALDSON E., NILAN R. A. and KONZAKC. F. The interaction of oxygen, radiation exposure and seed water content on y-irradiated barley seeds. ENVIRONMENTALAND EXPERIMENTAL BOTANY 19~ 153--164, 1979.--The roles of oxygen concentration, seed water content, and their interaction in the y-ray-induced damage to dry barley seeds were investigated. Himalaya (C.I. 620) barley seeds were adjusted to water contents ranging from 2 to 10(~,}), irradiated with 6°Co y-rays, and soaked at 0°C in distilled water bubbled with oxygennitrogen gas mixtures containing 0.0, 3.1, 6.25, 12.5, 25, 50 and 100% oxygen. Biological elt~cts of the treatments were recorded as M x seedling injury. Essentially no oxygen enhancement of biological damage was obtained with an oxygen concentration of 3.1 o~ in the gas phase of the soaking solution. The minimum OCHG needed to cause an oxygen enhancement of biological damage (3.1%OCHG) increased with increasing seed water content between 1.8 and 10.0~'0, and decreased as the radiation dose increased, suggesting a triple factor interaction. For moderate levels of injury (between 20 and 60% ), a nearly linear increase in seedling injury was obtained when the OCHG was increased in an exponential fashion. For greater seedling injury, the response tended toward a sigmoid shaped curve, probably due to limitations of the biological parameter. The same types of response were obtained when the results were analyzed in terms of oxygen enhancement ratios (OER) obtained from seedling injury data. Decreases in oxygen-independent radiosensitivity, determined by the increase in radiation exposure required to induce 40°o injury with anaerobic soakings, were Obtained as the seed water content was increased from 2.0 to 9.9%. The same pattern of radiosensitivity was observed with aerobic soakings and was more pronounced at intermediate levels of OCHG. The change in radiosensitivity of seeds between seed water contents of 2.0 and 7.7°:o was consistent with published EPR data. The increase in response at intermediate OCHG was in part a reflection of the influence of seed water content on the level of OCHG needed to produce an oxygen enhancement of damage. Cooling seeds of 6.1 °/o water content to dry ice temperatures immediately after irradiation at 0°C decreased both oxygen-dependent and oxygen-independent damage. The decreases in damage were greater at 20krad than at 10krad and tended to be greater at the higher OCHG. INTRODUCTION STUDIES in the past two decades have provided a wealth of knowledge about factors which
modify the increase in biological damage resulting from the reaction of oxygen with ionizing radiation products. M a n y of the studies were
*Scientific Paper No. 5109. College of Agriculture Research Center, Washington State University, Pullman, Project Nos. 1068 and 1568. Supported in part by a grant from the Department of Energy Contract No. EY76-S-06-2221. DOE RLO-2221-T2-39. Present address: Dry Land Research Unit, Lind, Washington 99341. 153
154
E. DONALDSON, R. A. NILAN and C. F. KONZAK
conducted on " d r y " systems such as organic macromolecules, bacteriophages, bacteria spores, insect eggs and plant seeds, and have provided new knowledge about the mechanisms by which ionizing radiation induces biological effects. Nevertheless, numerous gaps still exist in our understanding of these mechanisms, including those involving oxygen-dependent damage. It now appears that oxygen acts as a primary agent in reactions which lead to radiation damage in " d r y " systems such as seeds. O' 13, 24, 27, 28, 32, 34, 351 Water content, temperature and post-radiation storage appear to be secondary factors which may regulate the influence of oxygen on radiobiological damage. The extensive studies of CONGER et alJ121 and AHNSTROM and MIKAELSEN(11 have provided some understanding of the role of seed water content in the development of oxygendependent damage. COYOER et al. ~a2t observed that as the water content increases to a critical level, above which no post-irradiation oxygen enhancement of damage is obtainable, the magnitude of the oxygen-dependent damage is reduced. The oxygen enhancement (OE) of damage is not reduced upon storage of very dry seeds ( < 4 o, 'o) in a partial vacuum; however, storage of more moist seeds in a partial vacuum results in a decrease in OE. The rate of O E decrease in more moist (4-10°.{~) seeds increases with increasing seed water content. The E P R signal (reflecting the concentration of free radicals) follows this same pattern. Whenever E P R results have been compared with the results of oxygen-dependent damage occurring during soaking, a good correlation has been found, indicating that oxygen probably reacts with free radicals or oxygen sensitive sites (OSS) resulting from free radicals. The influence of temperature treatments applied post-radiation, ~7' 24, 28) during storage, O31 and during soaking ~0,2~1 has been investigated. High temperature post-radiation and storage treatments tend to decrease OE, possibly by loosening the three dimensional molecular structure of molecules near OSS and allowing for the formation of oxygen-unreactive configurations. It is possible that the critical water content for three dimensional structural changes depends on temperature: the higher the tem-
perature, the lower the critical water content. Increasing the temperature during soaking reduces the oxygen enhancement ratio (OER), (1°'211 possibly due at least in part to a lowering of the dissolved oxygen concentration. The influence of oxygen concentrations on the magnitude of radiation-induced damage in "wet" systems is quite well understood (z'3'23) even for oxygen tensions above that in air at atmospheric pressure. O7'391 The concentration of oxygen needed to realize an increase in biological damage with post-radiation soaking is still unknown, as is the response to increasing oxygen concentrations in the soaking medium. The objective of the present study was to investigate the interrelation of oxygen concentration, free radical concentration (irradiation exposure) and seed water content on the initiation of biological damage in the " d r y " system. In particular, the oxygen-dependent (free radical) dependent component of damage was investigated.
MATERIALS AND M E T H O D S
Preparation and treatment of biological material Medium sized (passed through 2.78x 19.05 mm and collected on 2.38 x 19.05 m m screen) seeds (caryopses) of the hulless barley (Hordeum vulgare), variety Himalaya (C.I. 620), were used in this .study. Seeds were brought to the desired water content (2-8°/~,) by desiccation over packets of calcium oxide in a partial vacuum. Seeds containing 10°,o water were obtained by storage over a glycerin water mixture in a vacuum desiccator. (13'25) Seeds were placed in 10mm diameter glass vials which were attached and removed from a glass manifold vacuum system with a glassworking torch. (2'21'321 The 100o water content seeds were actively pumped for at least 30rain and allowed to equilibrate on the system for at least 10hr after the last pumping before removal. Seeds containing 6-8°,0 water were actively pumped for at least 4hr, and those containing 2-4°o water were pumped for at least 10hr. The seed water contents reported in this study were measured (321 at least 10hr after removal from the system. ~6)
THE INTERACTION ON 7-IRRADIATED BARLEY SEEDS After irradiation the seeds were placed in perforated polyethylene vials which were immersed in distilled water at 0°C for postirradiation soaking. To achieve soaking solution atmosphere equilibrium, the distilled water was bubbled at least 0.5hr prior to seed soaking (16) and continuously during soaking with the appropriate gas (oxygen, nitrogen, or a mixture of these). Soaking at 0°C retards metabolic processes, provides the maximum concentration of dissolved oxygen and extends the development time for OE, (13) resulting in a higher O E R (21) than obtainable at higher temperatures. Unless otherwise stated, soaking was for at least 10hr, more than sufficient time for the reactions being studied to be completed at 0°C. (12) The oxygen and nitrogen gases used in this research were obtained in commercial gas cylinders. Nitrogen cylinders containing more than 0.1'!i~ oxygen were rejected.
Irradiation source and procedure The Washington State University 6°Co 7-ray facility in the nuclear reactor pool (4) was used, but with a new 6°Co loading (previously 2.1krad/min, now 15) and a new moveable radiation tube. In experiments with only dry seeds (=<6%) the vials of seeds were irradiated at room temperature while rotating at 6 0 r p m about their own and the radiation tube axis. When an experiment included seeds of 8 - 1 0 % water content, all vials of seeds were irradiated near 0°C in a Dewar flask while rotating about the carrier axis. Immediately after irradiation the vials were placed in an isopropyl alcoholdry ice mixture ( - 7 8 ° C ) and transported to the laboratory for soaking. (5)
Measurement of biological damage The soaked seeds were planted on blotter paper and the resultant seedlings cultured (26'31) for 5-6 days, at which time the length of the first seedling leaf was measured to the nearest millimeter. Three independent replications of 50 seeds each were used for each treatment. Dead seeds and seedlings showing mechanical damage were not included in the analyses. Mean seedling height for each treatment, coefficient of variation, and percent in-
155
jury (percentage reduction in M 1 seedling growth relative to the control) were calculated with the Seedling Height Program for the IBM 360 Computer of the Washington State University Computing Center. Analysis of variance for seedling height in factorial experiments was computed with the B M D O 8 V Program (is) from the treatment replication means. All other analyses were calculated by standard statistical methodsJ 3°' 30)
Proceduresfor individual experiments The objective of this investigation was to measure and determine the significance of interactions. Thus, a factorial design was used whenever practical. Two factorial experiments were used to study the trends, main effects, and interactions of seed water content, radiation dose, and O C H G . In one experiment the treatments were compared to a non-irradiated, nitrogen-soaked control of the same seed water content. The results are presented as percent reduction in seedling height. The second experiment made use of a comparison of exposures needed to obtain a 40o/0 reduction in seedling height with the same variables. The objective was to obtain a relationship of the radiosensitivities on a comparative basis.
Relation of OCHG to reduction in seedling height Two experiments were conducted to obtain the oxygen enhancement of damage over a relatively wide range of seed water content. Seeds at water contents of 1.8, 4.1 and 6.1°o were irradiated with 0, 5, 10, 15 and 20krad of 6°Co ?-rays while seeds of 6.1, 8.0 and 10.0°o water were treated with 0, 10, 20 and 30krad. In both experiments the seeds were soaked in water bubbled with O C H G of 0.0, 3.1, 6.25, 12.5, 25, 50 and 100%. Where the O C H G is doubled for each successive level, the dissolved oxygen is also doubled. At 0°C oxygen is soluble to 69.45ppm when the total pressure, 7 6 0 m m H g , is made up of oxygen and the aqueous tension, (22) 14.62 ppm when the total pressure is air (20.90o oxygen or 1 6 0 m m H g ) and the aqueous tension, ~3m and oxygen is virtually eliminated with
156
E. DONALDSON, R. A. NILAN and C. F. KONZAK
nitrogen bubbling (Beckman Model 777 laboratory oxygen analysis). From these data the approximate concentrations of dissolved oxygen for each O C H G can be calculated. They are: 0.0, 2.2, 4.3, 8.7, 17.3, 34.6 and 69.5ppm for O C H G of 0.0, 3.1, 6.25, 12.5, 25, 50 and 100°,~, respectively, at a total pressure of 760 m m H g at 0°C.
showed that the main effects of seed water content, irradiation and O C H G are all significant at the 99% confidence level. The seed water contentO C H G interaction and the triple interaction, seed water content-OCHG-radiation dose, in the experiment with the seeds of higher water content, are significant at the 90% confidence level. All other interactions are significant at the 99°J~, confidence level for both experiments. Relation of OCHG to doses giving the same biological The overall effect of seed water content (not effect illustrated) indicated a quadratic response with Seeds of 2.0, 4.3, 6.2, 7.7 and 9.9~).~ water the greatest difference occurring between seeds content were irradiated with 3 exposures of 1.8 and 4.1°/o water content. The radiosensiselected to estimate by graphing the exposure tivity of the seeds containing 6.1°/~ water was necessary to induce 40% injury when soaked at slightly less than that of the 4.1 °/o water content each O C H G . The oxygen enhancement ratios seeds, when averaged over all treatments. The (OER) were then calculated from: overall average seedling height of the seeds containing 8.0 and 10.0% water was the same. exposure required to produce The radiation response showed an apparent, 40% injury in nitrogen soaking" nearly linear main effect (not illustrated), with OER = a slight tendency toward a quadratic comexposure required to produce ponent for the higher seed water content experi40% injury in oxygen soaking ment and a very slight indication of a possible sigmoid or cubic component for the lower seed The O E R is usually defined as the ratio of water content experiment. With the levels on a exposures required to produce the same amount percentage basis and plotted on a log scale, the of biological damage, here specifically in seeds main effect (not illustrated) was apparently soaked, after irradiation, in nitrogen-saturated sigmoid with 3.1% oxygen having practically water relative to those soaked in oxygen- no effect and a nearly linear (exponential, due saturated water, but otherwise treated identi- to log scale) portion between 12.5 and 50~0 cally. For this experiment, in order to study the O C H G . The actual values as percent injury are change in O E R as the concentration of oxygen given in Table 1, where the average seedling is changed, O E R is redefined by replacing height of all nitrogen-soaked treatments within "oxygen-saturated water" with "water contain- an experiment were used as the control for that ing oxygen". The procedures followed in irra- range of seed water content. The values given diation and transfer to soaking were the same as are the average for each O C H G summed over those described for experiments containing seeds radiation dose and seed water content. Very of 10% water content. little effect occurred below 6.25% O C H G while over half of the effect was realized with 25°Jo O C H G . Nearly the entire OE was obtained RESULTS with 50% O C H G . Radiosensitivity measured by percent reduction in seedIn Figs. 1 and 2, the base indicates what ling height would be expected if radiation had no effect. If The influence of O C H G on the soaking OE the O C H G had no effect on the irradiated was analyzed with a factorial design so that the seeds, the resulting plane would be that :genesignificance of the interaction of the factors rated by the line for radiation doses at 0% could be demonstrated. An analysis of variance OCHG. on the M 1 seedling height for the 1.8, 4.1 and The OCHG-radiation dose interaction sur6.1 °,o seed water contents and that for the 6.1, faces are shown in Fig. 1 for the low seed water 8.0 and 10.0% seed water contents experiments content experiment. Since the three graphs look
THE INTERACTION ON 7-IRRADIATED BARLEY SEEDS
157
Table 1. Percent injury for irradiated baTley seed by oxygen concentration (°/o ) in the gas phase of the soaking solution summed over radiation exposure and seed water content
Seed water (o~)
Control* seed ht. (cm) 0",, O,
2-6 6-10
11.65 12.49
3.1
Oxygen concentration in the gas phase 6.25 12.5 25 ;l()
-0.5 1.4
2.5 4.4
14.0 11.7
23.9 22.0
100
33.6 31.7
39.3 38.4
*Average of three water contents.
J
l00
A. Z.SZ Seed ~ete~
~8/ ° - ' - - ~/"[
,o.,.
/
o7~-/~/~-/
1
krads
l
5
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I,o
7
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/16o
0
~
l°,e~-P~l d ~ d ~ f t
t8o .,-"7
oL ~ ~ ~ _ ~ ~ o 3.Z6.2 Z2 25 SO ZOO
20 o _ _ _ . ~ i f
Seed Water coutroZ xe.~ 6.1Z
15o;'/
k~.d. _ _ / /
0
3.1
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.
~o i16o l
~
140
i
// ~ / ~ / /°~ /
OCHG (p,,e,,,O
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J 100
o U/i,o .o v I
;. ,
_V,_----or/
6.2 12 25 50 OCllO (percent)
_d~.6
I
I
I
l
0
100
FIG. 1. Seedling injury response surfaces for oxygen concentration in the gas phase of the soaking solution (OCHG)-radiation dose interactions for "very dry" barley seeds.
E. DONALDSON, R. A. NILAN and C. F. KONZAK
158
100
A.
6 • I, Seed Water xx.86
--///
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,-,,
(per
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8.0% Seed Water C o n t r o l Helm
//
I=
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1
~o/--z'-//.///
OCHG
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OU
,oo
o
' 20
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/ /
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1
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3.1
6.2 OCHG
IO.OZ Seed Water Control Mean 13.44
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L.~I
~ ~'/
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,o ,.--,--¢ / M ' U ~ /-
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12 25 50 100 (perce.t)
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I
1 50
I
I 0
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l
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kr~ds'Z/~o/_J_.~o/~6/ 0
0
3.1
6 , 2 12 25 50 OCI~ ( p e r c e n t )
100
Fro. 2. Seedling injury response surfaces for oxygen concentration in the gas phase of the soaking solution (OCHG)-radiation dose interactions for "dry" barley seeds. nearly the same, the interactions with seed water content appear to be rather small. However, a close comparison of the radiation lines indicates that seeds of 4.1 and 6.1 (~'(~ water content react more alike at 5krad than do 1.8 and 4.1:'o water content seeds. At 15 and 20krad the radiation curves are very steep between 6.25 and 25°o O C H G on seeds of 1.8 and 4.1". water content, while on seeds of
6.1",, water content the slope is not as steep and continues to a higher O C H G . At 10krad seeds of 4.1°/0 water content showed a response more nearly centered between seeds of 1.8 and 6.1°/o/ water content. The seed water ContentO C H G interaction was the smallest of the double interactions and is easiest to see at the higher radiation doses and high O C H G , since seeds of 4.1 and 6.1 °/o water content responded
THE INTERACTION ON 7-IRRADIATED BARLEY SEEDS similarly, but seeds of 1.8'~, water content showed a tendency toward a q u a d r a t i c response. Between 6.25 and 12.5°,o O C H G seeds of 4.1°o water content responded more like seeds of 1.8~!/o water content. T h e surfaces themselves a m p l y demonstrate the great a m o u n t of interaction between O C H G and radiation dose. For each seed water content (Fig. 1 ), an arc can be d r a w n around the base of the start of this interaction. For example, the dotted line in Fig. 1C describes the arc for 6.1~Io seed water content in the lower water content experiment. As the seed water content is decreased, this arc moves toward a lower radiation dose with a lower O C H G . This same effect holds for seed water contents up to 10",o as is shown in Fig. 2. T h e response surfaces for O C H G - r a d i a t i o n exposure interaction for seeds of higher water content are shown in Fig. 2. T h e response due to seed water content was more nearly additive here than in Fig. 1. To compare these surfaces with those in the preceding figure, the u p p e r radiation level in this figure should be ignored. T h e injury on seeds of 6.1°~, water content resulting from 10 and 2 0 k r a d in this experiment was less than the injury ti~om similar treatment in the preceding experiment (Fig. 3). This difference must have been due to the difference in procedure used for the two experiments. T h e reduction in injury to these seeds would be due to cooling to - 7 8 ° C , or w a r m i n g back to 0~C. Neither should have 0 •
tO krads I0 krads; cooled t o - T B C
80
CI 20 kreds
70
159
caused a decrease in damage. ~st T h e change ill distance (° o injury) with a change in O C H G , between the lines for the respective radiation exposures, indicates a change in the m a g n i t u d e of the OE. T h e r e appears to be two components to the decrease in d a m a g e , one which effects the oxygen-independent d a m a g e and a second which influences the oxygend e p e n d e n t damage. T h e exact relationship could not be determined but there appears to be a small proportional decrease in d a m a g e with an increase in the radiation dose or in the m a g n i t u d e of the OE. T h e decrease was greater than would be expected from a strictlv additive response. T h e percentage of tile full oxygen enhancement (100~, o O C H G ) which was realized tbr each O C H G with each radiation exposure, summed over seed water content within each experiment, is shown in T a b l e 2. T h e values were calculated from the formula: Nitrogen S o a k i n g - - M i x e d Gas Soaking Nitrogen S o a k i n g I O x y g e n Soaking
x 100,
where each value was a seedling height in centimeters. In general, the higher the exposure of irradiation, the lower the O C H G must have been to show an oxygen enhancement or any portion of the full OE. A t 3.1°,o O C H G no consistent O E was obtained. An O C H G of 6.25°0 gave only a slight O E with the higher radiation exposures. Except for the low radiation exposures, about 25°o or more of the full O E was obtained with 12.5°~, O C H G , 5()~I~ or more with 2 5 ° ; O C H G , and 75%~, or more with 50~!o O C H G .
60 50 -
40
-~,
30
IO
O0
5I
6.25
02 Concentration
12 5 in t h e
25 SoaKing
50
o
I00
Gas (%)
FIG. 3. Effect of controlled (irradiated at 0°C and cooled to - 7 8 C) and uncontrolled temperature during irradiation and pri~w to soaking on the ~ccdlin~ i,,im> r,'Sl),ms," ,,l'l~,u'h'\ scotia. B
Radiosensitivity measured by a comparison of doses giving the same biological effect T h e radiation doses required to give 40°o injury to seeds at each of the five seed water contents soaked with each of the levels of O C H G are shown in Fig. 4. All of the O C H G curves indicate that there m a y have been a q u a d r a t i c c o m p o n e n t for seed water content. T h e q u a d r a t i c response for any one line m a y not be significant, but since this same response was obtained for the 7 soaking atmospheres,
160
E. DONALDSON, R. A. NILAN and C. F. KONZAK
Table 2. Percent oJ" the Jull oxygen enhancement obtained Jor each exposure of radiation used in barley seeds, summed over seed water content Oxygen conc. in the gas phase Seed water content 1",, )
Exposure (krad)
N 2 hyd* (cm)
5 10 15 20 10 20 30
11.51 11.34 10.55 10.47 12.68 12.57 11.84
2q5
6-10
3.1 6.25 12.5 25 50 Percent of full oxygen enhancement - 13.0 3.5 -2.7 0.7 3.8 5.6 1.4
1.5 12.2 2.6 10.0 2.5 10.2 14.0
23.6 36.7 26.6 43.7 19.4 25.9 38.0
34.2 62.5 52.3 68.6 41.3 46.5 70.1
79.4 84.5 81.5 90.0 73.5 74.5 90.4
Full Oz"~ enhancement (cm) 1.99 5.10 7.05 8.12 2.37 7.49 9.30
*Seedling height (cm) of the sample soaked in niu'ogen-bubblcd water. Average of 3 replications. ' F u l l oxygen enhancement as the difference het~een tile seedling height of the samples soaked in nitrogen and those soaked in 100t!i, oxygen.
~_~6
0
~
7
0 0.0%OCHG
50
625%0CHG
L~J
I0 ~
I~50CHG
C
H
G
SeedWaterContent(%) Fro. 4. Radiation doses required to give 40°0 injury to barley seeds of 5 water contents when soaked in each of 7 oxygen concentrations in the gas phase of the soaking solution (OCHG).
each line having been obtained from the average of three replications, the evidence was quite strong that, for the procedure used, the quadratic response did exist. T h e greatest change occurred between 2.0 and 4.3% seed water contents, with a more gra~tual change between seeds of 4.3 and 7.7°:o water content, followed
by a negative slope between seed water contents of 7.7 and 9.9~I0. The m a x i m u m change or reduction in radiosensitivity occurred between seeds of 2.0 and 7.7°:o water content and was greatest at 6.253o O C H G , being 14.7krad. T h e greater radiosensitivity of seeds of 9.9')o water content c o m p a r e d to seeds of 7.7~{~ water content was very consistent, ranging from a m a x i m u m of 4.Tkrad at 3.1°,b O C H G to 0 . 5 k r a d at 5 0 ° ' 0 0 C H G . At 100°o O C H G , seeds of 9.9".~, water content showed about the same level of radiosensitivity as seeds of 7.7~'o water content. A comparison of the 8.0 and 10.0°i, water content seed in graphs B and C in Fig. 2 shows that the 10.0°0 water content seeds have a greater radiosensitivity at intermediate O C H G than the 8.0°./0 water content seeds. T h e distance between the lines, Fig. 4, indicates the change in oxygen-dependent damage ( r a d i a t i o n - o x y g e n interaction) associated with the change in O C H G . Since the lines for 0.0 and 3.1~!{) O C H G and those for 25, 50 and l(/0°o O C H G were much closer together than those for the intermediate O C H G ' s , a sigmoid curve or cubic response was indicated. In Fig. 5 the base indicates an O E R of 1.0, as obtained with either a very low oxygen concentration or with a seed water content somewhat above 10 °':0" T h e area between the
THE INTERACTION ON 7-IRRADIATED BARLEY SEEDS
161
attempted to study different concentrations by mixing oxygenated water with oxygen-free water. With very dry seeds a mixture containing as little as 1°{~ oxygenated water gave 30 and 650o of the maximum oxygen effect with 10 and 20krad, respectively. One of the pro~5 blems with this system was its unsuitability for g determining the concentration of oxygen in the gas phase of the soaking solution (OCHG). For % Seed 2.~. instance, the actual dissolved oxygen conWater / centration for a mixture containing 1° o C°nfent6~ oxygenated water was not equivalent to 7.7j---"--'~7.7 1°~ O C H G but was 10.1°..o O C H G . Where the soaking water was continuously bubbled 0 3.1 6.25 12.5 25 ' ;o ,oo with various concentrations of oxygen in the gas 0 z Concentration in the Sooking Gas (%) FIG. 5. Oxygen enhancement ratio (OER) response phase, about 25, 50 and 80°o of the maximum surface fi)r oxygen concentration in the gas phase of oxygen enhancement (OE) was realized with respectively the soaking solution (OCHG)-barley seed water con- 12.5, 25 and 50c),,~ O C H G , (Table 2). Seed water content and radiation tent interactions. exposure both have an influence on the results. reaction surface and the expected surface (dot- The values published earlier (17) were slightly ted line) was the seed water c o n t e n t - O C H G higher than those reported here, but were averinteraction for this experiment. The area be- aged over radiation exposure rather than seed tween the base and the expected surface was water content and were for seeds of less than the O E for 10°~ water content seeds. This 6°o water content. The results from 'Fable 2 indicate that in the figure gives essentially the same information as " d r y " system the range of oxygen concentration Figs. 1 and 2, only in a different form. At the required to produce the O E during posthi~her seed water contents, namely 6.2, 7.7 radiation soaking is probably both broader and and 9.9(Io, the O E R values showed an apparent linear response from 6.25 to 100°'o O C H G when higher than the range required during irradiation in the "wet" system. An O C H G of over O C H G was plotted on a log scale. 3.1°.o is required for a consistent O E in irradiated barley seeds. The present study with DISCUSSION A N D CONCLUSIONS barley seeds does not establish an upper limit The purpose of varying the O C H G was to for the oxygen concentration influence. regulate the partial pressure of oxygen in the However, the need for an exponential increase atmosphere in equilibrium with the soaking in O C H G to obtain a linear increase in seedling water. The c~,mcntration of dissolved oxygen is injury indicates that a very large increase in the directly related to the partial pressure of oxygen dissolved oxygen concentration might be reif all other factors are held constant. The other quired to attain the level of injury realized with factors having the greatest influence on the irradiation of seeds in oxygen at high concentration of dissolved oxygen are tempera- pressures.(16) ture, dissolved salts or soluble organic comThe influence of seed water content on the pounds and barometric pressure. These would oxygen-dependent and independent damage ocnot have changed much during the course of an curring during soaking has been thoroughly reviewed {11'12) and will be discussed here only experiment, due to the methods used. In earlier reports, the effects of oxygen con- where relevant to this study. The radiosensicentration in the gas used for seed soaking was tivity of seeds soaked in oxygen-free water alexamined at three levels, 100, 20.9 (air) and tered little between seed water contents of 2.0 0",, Ils'~9'es~ using "'dry'" seeds. NIl.AN et al. t3"*) and 6.20~,, but decreased at 7.7°0 water (Fig. 3). a ._o
'
162
E. DONALI)SON, R. A. NIl,AN and C. F. KONZAK
This is in agreement with the results of CONOER et al. (~2) As the oxygen concentration increased
(logarithmically), radiosensitivity became almost linear at 1 2 . 5 ° / 0 0 C H G and remained linear with practically no change in magnitude up to 100% O C H G . The radiosensitivity followed the radical signal amplitude found by CONCER,(9) decreasing with increasing water content and was probably a simple reflection of this change in radical signal amplitude. The maximum injury was obtained at the lower seed water content, 2.0~I~,, which is similar to CONOER'S{8) results but differs slightly from those of CALDECOTT~6) and CONGER el al. ~x2) CALDECOTT(6) and CONGER el a/. ~12) obtained maximum seed radiosensitivity between 3 and 4°0 water content. Part of the difference in which seed water content showed the maximum radiosensitivity could be due to the method of presenting the results. The greatest interaction resulted from the difference between the i n j u r y response to O C H G of 1.8°o water content seeds and those of 4.1 and 6.10;0 seeds (Figs. 1, 2, 4 and 5). The influence is greatest at intermediate oxygen concentrations, which were not used in other investigations. The most striking effect of water content was greater radiosensitivity of the 10°o water content seeds. The reason for greater radiosensitivity of 10% water content seeds compared to 8 ', O water content seeds is not understood 'but could be due to the change in binding characteristics of water near the seed water content where the post-radiation OE ceases to occur. Seed water content interacts with oxygen concentration in at least two ways. As already noted, increasing the seed water content decreases the magnitude of the OE. ~x2'33) The other interaction involves the concentration of oxygen required to show an OE. As the seed water content is increased, the oxygen concenlration must also increase (Figs. 1 and 2) to obtain the same level of damage. As radiation exposure is decreased, the oxygen concentration must be increased to obtain the same level of damage. A triple interaction seems to occur. The interaction between radiation dose and oxygen concentration in the soaking gas is the most significant of the interactions studied in the present investigation. This interaction re-
flects the influence of oxygen concentration on the attainment of potential OE for the different exposures of radiation studied. Graphs of oxygen concentration vs percent injury provide a family of curves which have slopes increasing in steepness with an increase in oxygen concentration (Figs. 1, 2 and 5). A similar relationship is obtained by irradiating bacterial cells with high-intensity-pulsed electrons in the presence of increasing oxygen concentrations. (2°) In bacteria, the effect can be explained (14) by assuming that the first few krad of exposure deplete the oxygen within a cell by radiationinduced chemical reactions. The exposure required to deplete the oxygen by reaction would depend exclusively on the oxygen concentration. The rest of the exposure would be given in essentially anoxic conditions. The time for the soaking oxygen effect in seeds may be limited under certain conditions and the extent of reaction depends both on the oxygen concentration and the radiation exposure (16) as in the bacterial system. In the soaking oxygen enhancement, the level of effect depends on the oxygen diffused into the cells (of barley seeds), not the oxygen present at the time of irradiation. Since the magnitude of the reaction (Figs. 1, 2 and 5 show the completed reactions) is dependent on the radiation exposure and the time required for the elimination of oxygen sensitive sites (OSS), the oxygen-OSS reaction rate is dependent on OSS and oxygen concentrations, ~16) and interaction is the natural result. The decrease in damage due to the irradiation of seeds at 0°C and cooling to - 7 8 ° C (Fig. 3) was not expected and consequently was investigated only on seeds of 6°/o water content. The procedure was used as a convenience to prevent the decay of radicals in the more moist seeds during their transfer to the laboratory for soaking and to take advantage of the greater potential oxygen enhancement obtained from irradiating seeds at temperatures between 0 and 20°C as found by KRAtJSSE.~29) The reduction in damage due to immediate post-irradiation cooling of seeds is not the same as that resulting from the post-radiation heatshock used by KONZAK et a1328) and NIl.AN et a l / 3 e l The post-radiation heat-shock eliminated
THE INTERACTION ON ~,-IRRADIATED BARLEY SEEDS the soaking O E , gave a higher m u t a t i o n ti'equency with less chromosomal d a m a g e , and gave very little change in the seedling height. W h e n X - i r r a d i a t e d seeds were m a i n t a i n e d at 75-85°C for more than 4 8 h r , the d a m a g e decreased with an elimination of the oxygen enh a n c e m e n t ) 7) KESAVAN and KAMRA(24) found a similar response for heat-shock and microwaves. T h e elimination of the soaking oxygen enhancem e n t seems to be the major means of " r e d u c ing" d a m a g e to seeds in these experiments. T h e reduction in d a m a g e resulting from the i m m e d i a t e post-radiation cooling of seeds has its major influence on the o x y g e n - i n d e p e n d e n t damage. T h e effect of post-irradiation cooling on the o x y g e n - d e p e n d e n t d a m a g e is m i n i m a l and would p r o b a b l y be considered as part of the error, as BOTTINO(5) has done, if observed on only one point. T h e reduction in oxygend e p e n d e n t d a m a g e was greater for 2 0 k r a d than for 10krad and tended to be greatest for oxygen concentrations showing the most o x y g e n - d e p e n d e n t d a m a g e . CALDECOTT(7) obtained a similar reduction in the oxygend e p e n d e n t d a m a g e by heating seeds to 75°C for 2 5 h r prior to irradiation. H e a t i n g prior to irradiation could have a pre-conditioning effect on the possible sites for radicals, making them less receptive and resulting in less d a m a g e . This could not explain the post-radiation cooling effect. Both effects could be explained, however, by a change in the molecular structure accomp a n y i n g heat treatments or associated with a change in t e m p e r a t u r e , which would allow the harmless dissipation of a portion of the radiation energy absorbed. This change in molecular structure could be a shift in the three dimensional configuration of macromolecules moving reactive sites closer together or farther apart.
REFERENCES 1. AHNSTROM G. and MIKAELSEN K. (1968) Lifetimes of radiation-induced oxygen-sensitive centres in barley embryos soaked at different temperatures. Pages 283-286 in Mutations in plant breeding II, International Atomic Energy Agency, Vienna.
163
2. ALPER T. and MOORE J. L. (1967) The interdependence of oxygen enhancement ratios for 250 kVp X-rays and fast neutrons. Br. J. Radiol. 410, 843-848. 3. BAKER W. K. (1955) The oxygen effect and the mutation process. Brookhaven Symp. Biol. 8, 191200. 4. BOTCnEK C. M., KONZAKC. F. and NILAN R. A. (1964) Design of the 6°Co facility at Washington State University. IEEE Tram'. Prod. Eng. Prod.
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. CALDECOTT R. S. (1955') Effects of hydration oil X-ray sensitivity in Hordeum. Radiat. Res. 3~ 316 330. 7. CALDECOTTR. S. (1961) Seedling height, oxygen availability, storage and temperature: Their relation to radiation-induced genetic and seedling injury in barley. Pages 3-24 in Effects of ionizing radiation on seeds, International Atomic Energy Agency, Vienna. 8. CONGERA. 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, Publication 1355, Washington, D.C. 9. CONGERB. V. (1967} The influence of seed water content on the oxygen effect in irradiated barley seeds, Ph.D. Thesis, Washington State University. 91 p. 10. CONGER B. V., HILEMANJ. R., NILAN R. A. and KONZAK C. F. (1971)The influence of temperature on radiation-induced oxygen-dependent and -independent damage in barley seeds. Radiat. Res. 469 601~512. 11. CONGER B. V., NILAN R. A. and KONZAK C. F. (1968) Post-irradiation oxygen sensitivity of barley seeds varying slightly in water content. Radiat. Bot. 8~ 31 36. 12. CONGER B. V., NILAN R. A. and KONZAK C. F. (1969) The role of water content in the decay of radiation-induced oxygen-sensitive sites in barley seeds during post-irradiation hydration. Radiat. Res. 399 45 56. 13. CONGER B. V., NILAN 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. 69 129-144. 14. DEWEY D. L. and BOAG J. W. (1959) Modification of the oxygen effect when bacteria are given large pulses of radiation. .~ature (London) 183, 1450-1451.
164
1". I)()N,\IA)St)N. R . . \ . NIl,AN and (',. 1:. K()NZ.\K
15. DIxoN W. J. (1964) Analysis of variance, BMDO8V. In Biomedical computer programs, Health Services Computing Facility, University of California, Los Angeles. 16. DONALDSON E. (1970) The interaction of some factors influencing the oxygen effect in irradiated barley seeds. Ph.D. 'Thesis, Washington State University. 114 p. 17. DONALDSONE., .~ILAN R. A. and KONZAK C. F. (1968) The inlltlcnce of oxygen concentration on the post-irradiation oxygen effect in barley seeds. Barley Newsl. 11, 34-35. 18. EHRENBERGL. (1955) Studies on the mechanism of action of ionizing radiations in plant seeds. Sven. Kem. Tidskr. 67, 207-224. 19. EHRENBERG g. (1955) The radiation induced growth inhibition in seedlings. Bot. Not. 1118, 184215. 20. EPP E. R., WEiss H. and SANTOMASSOA. (1968) The oxygen effect in bacterial cells irradiated with high-intensity pulsed electrons. Radiat. Res. 3t, 320-325. 21. HARLEJ. R. (1963) Modification of radiobiological oxygen effects in very dry barley seeds. Ph.D. Thesis, Washington State University. 68 p. 22. HODGMEN C. D., ed. (1949) Handbook of chemistTy and physics. Chemical Rubber Publishing Co,, Cleveland. 23. HOWARD--FLANDERS P. and ALPER T. (1957) The sensitivity of micro-organisms to irradiation under controlled gas conditions. Radiat. Res. 7, 518-540. 24. KESAVANP. C. and KAMRAO. P. (1971) Studies on the postradiation modification of the gammaray induced damage in barley seeds by heatshock and microwaves. Pages 127 137 in R. A. NILAN, ed. Barley genetics II, Washington State University Press, Pullman. 25. KONZAKC. F., BOTTINOP. J., NILAN 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 see& II, International Atomic Energy A ffemx. Vienna. 26. KONZAK C. F., MIKAELSEN K., SIGURBJ6RN~SO:,; B. and BURTSCHERA. (1967) Recommended standard procedures for irradiating, cultivating and measuring cereal seeds to determine the effects of neutron irradiation in the neutron-seedirradiation programme. Pages 103-1(17 in Neutron irradiation of seeds, Technical Reports Series No. 76, International Atomic Energx Agency, Vienna.
27. KONZAK C. F., N1LAN R. A., HARLE J. R. and HEINER R. E. (1969) Control of factors affecting the response of plants to mutagens. Brookhaven Symp. Biol. 1t, 128-157. 28. KONZAKC. F., NILAN R. A., LECAVLTR. R. and HEINER R. E. (1961) Modification of induced genetic damage in seeds. Pages 155-169 in Effects of ionizing radiation on seeds, International Atomic Energy Agency, Vienna. 29. KRAUSSEG. W. (1968) Possibility of influencing radiosensitivity and mutability in barley. III. The combined effect of X-rays and various levels of temperature during irradiation and the influence of humidity and the stage of germination of the caryopses. Z. Pflanzenziichtg. 60, 19M,0. 30. LI C. C. (1964) Introduction to experimental statistics. McGraw-Hill, New York. 460 p. 31. MYHILL R. R. and KONZAKC. F. (1967) A new technique for culturing and measuring barley seedlings. Crop Sci. 7, 275 276. 32. NmAN R. A., 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. Pages 171-182 in Strahlenwirkung und milieu (Suppl. to Strahlentherapie, Vol. 51 ), Urban & Schwarzenberg, Munich. 33. NmAN R. A., KONZAK C. F., HARLE J. R. and LE~AtJLT R. R. (1963) The magnitude of the oxygen effect in irradiated barley seeds. Page 94 in S. J. GEERTS, ed. Genetics to@, Vol. 1, Pergamon Press, Oxford. 34. Nn,AN R. A., KONZAKC. F., LEGAUUrR. R. and HARLE J. R. (1961) The oxygen effect in barley seeds. Pages 139-154 in Effects of ionizing radiations on seeds, International Atomic Energy Agency, Vienna. 35. SPARRMANB., EHRENBERGL. and EHRENBERGA. (1959) Scavenging of free radicals and radiation protection by nitric oxide in plant seeds. Acta Chem. Scand. 13, 199-200. 36. STF.EL R. G. D. and TORRIE J. H. (1960) tSineiples and plocedures of statistics. McGraw-Hill, New York. 481 p. 37. THOMLINSON R. H. (1964) Treatment of animal tumors. Br. J. Radiol. 37, 720 (Abstract). 38. WASSERA. (1965) Beckma n Instructions 1223-C. Beckman Instruments, Inc., Fullerton, CA. 39. WITHERS R. and SCOTT O. C. A. (1964) 'The oxygen effect in normal tissue. Br. J. Radiol, 37, 720 (Abstract).
0098-8472/79/0801-0153 $02.00/0
c; Pert~amon Press Ltd. 1979. Printed in Great Britain
THE INTERACTION OF OXYGEN, RADIATION EXPOSURE AND SEED WATER CONTENT ON 7-IRRADIATED BARLEY SEEDS* E. DONAI.I~SON, R. 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 13 June 1978; in revisedform 9 November 1978; accepted 13 November 1978) DONALDSON E., NILAN R. A. and KONZAKC. F. The interaction of oxygen, radiation exposure and seed water content on y-irradiated barley seeds. ENVIRONMENTALAND EXPERIMENTAL BOTANY 19~ 153--164, 1979.--The roles of oxygen concentration, seed water content, and their interaction in the y-ray-induced damage to dry barley seeds were investigated. Himalaya (C.I. 620) barley seeds were adjusted to water contents ranging from 2 to 10(~,}), irradiated with 6°Co y-rays, and soaked at 0°C in distilled water bubbled with oxygennitrogen gas mixtures containing 0.0, 3.1, 6.25, 12.5, 25, 50 and 100% oxygen. Biological elt~cts of the treatments were recorded as M x seedling injury. Essentially no oxygen enhancement of biological damage was obtained with an oxygen concentration of 3.1 o~ in the gas phase of the soaking solution. The minimum OCHG needed to cause an oxygen enhancement of biological damage (3.1%OCHG) increased with increasing seed water content between 1.8 and 10.0~'0, and decreased as the radiation dose increased, suggesting a triple factor interaction. For moderate levels of injury (between 20 and 60% ), a nearly linear increase in seedling injury was obtained when the OCHG was increased in an exponential fashion. For greater seedling injury, the response tended toward a sigmoid shaped curve, probably due to limitations of the biological parameter. The same types of response were obtained when the results were analyzed in terms of oxygen enhancement ratios (OER) obtained from seedling injury data. Decreases in oxygen-independent radiosensitivity, determined by the increase in radiation exposure required to induce 40°o injury with anaerobic soakings, were Obtained as the seed water content was increased from 2.0 to 9.9%. The same pattern of radiosensitivity was observed with aerobic soakings and was more pronounced at intermediate levels of OCHG. The change in radiosensitivity of seeds between seed water contents of 2.0 and 7.7°:o was consistent with published EPR data. The increase in response at intermediate OCHG was in part a reflection of the influence of seed water content on the level of OCHG needed to produce an oxygen enhancement of damage. Cooling seeds of 6.1 °/o water content to dry ice temperatures immediately after irradiation at 0°C decreased both oxygen-dependent and oxygen-independent damage. The decreases in damage were greater at 20krad than at 10krad and tended to be greater at the higher OCHG. INTRODUCTION STUDIES in the past two decades have provided a wealth of knowledge about factors which
modify the increase in biological damage resulting from the reaction of oxygen with ionizing radiation products. M a n y of the studies were
*Scientific Paper No. 5109. College of Agriculture Research Center, Washington State University, Pullman, Project Nos. 1068 and 1568. Supported in part by a grant from the Department of Energy Contract No. EY76-S-06-2221. DOE RLO-2221-T2-39. Present address: Dry Land Research Unit, Lind, Washington 99341. 153
154
E. DONALDSON, R. A. NILAN and C. F. KONZAK
conducted on " d r y " systems such as organic macromolecules, bacteriophages, bacteria spores, insect eggs and plant seeds, and have provided new knowledge about the mechanisms by which ionizing radiation induces biological effects. Nevertheless, numerous gaps still exist in our understanding of these mechanisms, including those involving oxygen-dependent damage. It now appears that oxygen acts as a primary agent in reactions which lead to radiation damage in " d r y " systems such as seeds. O' 13, 24, 27, 28, 32, 34, 351 Water content, temperature and post-radiation storage appear to be secondary factors which may regulate the influence of oxygen on radiobiological damage. The extensive studies of CONGER et alJ121 and AHNSTROM and MIKAELSEN(11 have provided some understanding of the role of seed water content in the development of oxygendependent damage. COYOER et al. ~a2t observed that as the water content increases to a critical level, above which no post-irradiation oxygen enhancement of damage is obtainable, the magnitude of the oxygen-dependent damage is reduced. The oxygen enhancement (OE) of damage is not reduced upon storage of very dry seeds ( < 4 o, 'o) in a partial vacuum; however, storage of more moist seeds in a partial vacuum results in a decrease in OE. The rate of O E decrease in more moist (4-10°.{~) seeds increases with increasing seed water content. The E P R signal (reflecting the concentration of free radicals) follows this same pattern. Whenever E P R results have been compared with the results of oxygen-dependent damage occurring during soaking, a good correlation has been found, indicating that oxygen probably reacts with free radicals or oxygen sensitive sites (OSS) resulting from free radicals. The influence of temperature treatments applied post-radiation, ~7' 24, 28) during storage, O31 and during soaking ~0,2~1 has been investigated. High temperature post-radiation and storage treatments tend to decrease OE, possibly by loosening the three dimensional molecular structure of molecules near OSS and allowing for the formation of oxygen-unreactive configurations. It is possible that the critical water content for three dimensional structural changes depends on temperature: the higher the tem-
perature, the lower the critical water content. Increasing the temperature during soaking reduces the oxygen enhancement ratio (OER), (1°'211 possibly due at least in part to a lowering of the dissolved oxygen concentration. The influence of oxygen concentrations on the magnitude of radiation-induced damage in "wet" systems is quite well understood (z'3'23) even for oxygen tensions above that in air at atmospheric pressure. O7'391 The concentration of oxygen needed to realize an increase in biological damage with post-radiation soaking is still unknown, as is the response to increasing oxygen concentrations in the soaking medium. The objective of the present study was to investigate the interrelation of oxygen concentration, free radical concentration (irradiation exposure) and seed water content on the initiation of biological damage in the " d r y " system. In particular, the oxygen-dependent (free radical) dependent component of damage was investigated.
MATERIALS AND M E T H O D S
Preparation and treatment of biological material Medium sized (passed through 2.78x 19.05 mm and collected on 2.38 x 19.05 m m screen) seeds (caryopses) of the hulless barley (Hordeum vulgare), variety Himalaya (C.I. 620), were used in this .study. Seeds were brought to the desired water content (2-8°/~,) by desiccation over packets of calcium oxide in a partial vacuum. Seeds containing 10°,o water were obtained by storage over a glycerin water mixture in a vacuum desiccator. (13'25) Seeds were placed in 10mm diameter glass vials which were attached and removed from a glass manifold vacuum system with a glassworking torch. (2'21'321 The 100o water content seeds were actively pumped for at least 30rain and allowed to equilibrate on the system for at least 10hr after the last pumping before removal. Seeds containing 6-8°,0 water were actively pumped for at least 4hr, and those containing 2-4°o water were pumped for at least 10hr. The seed water contents reported in this study were measured (321 at least 10hr after removal from the system. ~6)
THE INTERACTION ON 7-IRRADIATED BARLEY SEEDS After irradiation the seeds were placed in perforated polyethylene vials which were immersed in distilled water at 0°C for postirradiation soaking. To achieve soaking solution atmosphere equilibrium, the distilled water was bubbled at least 0.5hr prior to seed soaking (16) and continuously during soaking with the appropriate gas (oxygen, nitrogen, or a mixture of these). Soaking at 0°C retards metabolic processes, provides the maximum concentration of dissolved oxygen and extends the development time for OE, (13) resulting in a higher O E R (21) than obtainable at higher temperatures. Unless otherwise stated, soaking was for at least 10hr, more than sufficient time for the reactions being studied to be completed at 0°C. (12) The oxygen and nitrogen gases used in this research were obtained in commercial gas cylinders. Nitrogen cylinders containing more than 0.1'!i~ oxygen were rejected.
Irradiation source and procedure The Washington State University 6°Co 7-ray facility in the nuclear reactor pool (4) was used, but with a new 6°Co loading (previously 2.1krad/min, now 15) and a new moveable radiation tube. In experiments with only dry seeds (=<6%) the vials of seeds were irradiated at room temperature while rotating at 6 0 r p m about their own and the radiation tube axis. When an experiment included seeds of 8 - 1 0 % water content, all vials of seeds were irradiated near 0°C in a Dewar flask while rotating about the carrier axis. Immediately after irradiation the vials were placed in an isopropyl alcoholdry ice mixture ( - 7 8 ° C ) and transported to the laboratory for soaking. (5)
Measurement of biological damage The soaked seeds were planted on blotter paper and the resultant seedlings cultured (26'31) for 5-6 days, at which time the length of the first seedling leaf was measured to the nearest millimeter. Three independent replications of 50 seeds each were used for each treatment. Dead seeds and seedlings showing mechanical damage were not included in the analyses. Mean seedling height for each treatment, coefficient of variation, and percent in-
155
jury (percentage reduction in M 1 seedling growth relative to the control) were calculated with the Seedling Height Program for the IBM 360 Computer of the Washington State University Computing Center. Analysis of variance for seedling height in factorial experiments was computed with the B M D O 8 V Program (is) from the treatment replication means. All other analyses were calculated by standard statistical methodsJ 3°' 30)
Proceduresfor individual experiments The objective of this investigation was to measure and determine the significance of interactions. Thus, a factorial design was used whenever practical. Two factorial experiments were used to study the trends, main effects, and interactions of seed water content, radiation dose, and O C H G . In one experiment the treatments were compared to a non-irradiated, nitrogen-soaked control of the same seed water content. The results are presented as percent reduction in seedling height. The second experiment made use of a comparison of exposures needed to obtain a 40o/0 reduction in seedling height with the same variables. The objective was to obtain a relationship of the radiosensitivities on a comparative basis.
Relation of OCHG to reduction in seedling height Two experiments were conducted to obtain the oxygen enhancement of damage over a relatively wide range of seed water content. Seeds at water contents of 1.8, 4.1 and 6.1°o were irradiated with 0, 5, 10, 15 and 20krad of 6°Co ?-rays while seeds of 6.1, 8.0 and 10.0°o water were treated with 0, 10, 20 and 30krad. In both experiments the seeds were soaked in water bubbled with O C H G of 0.0, 3.1, 6.25, 12.5, 25, 50 and 100%. Where the O C H G is doubled for each successive level, the dissolved oxygen is also doubled. At 0°C oxygen is soluble to 69.45ppm when the total pressure, 7 6 0 m m H g , is made up of oxygen and the aqueous tension, (22) 14.62 ppm when the total pressure is air (20.90o oxygen or 1 6 0 m m H g ) and the aqueous tension, ~3m and oxygen is virtually eliminated with
156
E. DONALDSON, R. A. NILAN and C. F. KONZAK
nitrogen bubbling (Beckman Model 777 laboratory oxygen analysis). From these data the approximate concentrations of dissolved oxygen for each O C H G can be calculated. They are: 0.0, 2.2, 4.3, 8.7, 17.3, 34.6 and 69.5ppm for O C H G of 0.0, 3.1, 6.25, 12.5, 25, 50 and 100°,~, respectively, at a total pressure of 760 m m H g at 0°C.
showed that the main effects of seed water content, irradiation and O C H G are all significant at the 99% confidence level. The seed water contentO C H G interaction and the triple interaction, seed water content-OCHG-radiation dose, in the experiment with the seeds of higher water content, are significant at the 90% confidence level. All other interactions are significant at the 99°J~, confidence level for both experiments. Relation of OCHG to doses giving the same biological The overall effect of seed water content (not effect illustrated) indicated a quadratic response with Seeds of 2.0, 4.3, 6.2, 7.7 and 9.9~).~ water the greatest difference occurring between seeds content were irradiated with 3 exposures of 1.8 and 4.1°/o water content. The radiosensiselected to estimate by graphing the exposure tivity of the seeds containing 6.1°/~ water was necessary to induce 40% injury when soaked at slightly less than that of the 4.1 °/o water content each O C H G . The oxygen enhancement ratios seeds, when averaged over all treatments. The (OER) were then calculated from: overall average seedling height of the seeds containing 8.0 and 10.0% water was the same. exposure required to produce The radiation response showed an apparent, 40% injury in nitrogen soaking" nearly linear main effect (not illustrated), with OER = a slight tendency toward a quadratic comexposure required to produce ponent for the higher seed water content experi40% injury in oxygen soaking ment and a very slight indication of a possible sigmoid or cubic component for the lower seed The O E R is usually defined as the ratio of water content experiment. With the levels on a exposures required to produce the same amount percentage basis and plotted on a log scale, the of biological damage, here specifically in seeds main effect (not illustrated) was apparently soaked, after irradiation, in nitrogen-saturated sigmoid with 3.1% oxygen having practically water relative to those soaked in oxygen- no effect and a nearly linear (exponential, due saturated water, but otherwise treated identi- to log scale) portion between 12.5 and 50~0 cally. For this experiment, in order to study the O C H G . The actual values as percent injury are change in O E R as the concentration of oxygen given in Table 1, where the average seedling is changed, O E R is redefined by replacing height of all nitrogen-soaked treatments within "oxygen-saturated water" with "water contain- an experiment were used as the control for that ing oxygen". The procedures followed in irra- range of seed water content. The values given diation and transfer to soaking were the same as are the average for each O C H G summed over those described for experiments containing seeds radiation dose and seed water content. Very of 10% water content. little effect occurred below 6.25% O C H G while over half of the effect was realized with 25°Jo O C H G . Nearly the entire OE was obtained RESULTS with 50% O C H G . Radiosensitivity measured by percent reduction in seedIn Figs. 1 and 2, the base indicates what ling height would be expected if radiation had no effect. If The influence of O C H G on the soaking OE the O C H G had no effect on the irradiated was analyzed with a factorial design so that the seeds, the resulting plane would be that :genesignificance of the interaction of the factors rated by the line for radiation doses at 0% could be demonstrated. An analysis of variance OCHG. on the M 1 seedling height for the 1.8, 4.1 and The OCHG-radiation dose interaction sur6.1 °,o seed water contents and that for the 6.1, faces are shown in Fig. 1 for the low seed water 8.0 and 10.0% seed water contents experiments content experiment. Since the three graphs look
THE INTERACTION ON 7-IRRADIATED BARLEY SEEDS
157
Table 1. Percent injury for irradiated baTley seed by oxygen concentration (°/o ) in the gas phase of the soaking solution summed over radiation exposure and seed water content
Seed water (o~)
Control* seed ht. (cm) 0",, O,
2-6 6-10
11.65 12.49
3.1
Oxygen concentration in the gas phase 6.25 12.5 25 ;l()
-0.5 1.4
2.5 4.4
14.0 11.7
23.9 22.0
100
33.6 31.7
39.3 38.4
*Average of three water contents.
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E. DONALDSON, R. A. NILAN and C. F. KONZAK
158
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/
,o
/ 1t° / ~
,o ,.--,--¢ / M ' U ~ /-
C.
.
12 25 50 100 (perce.t)
[
I
1 50
I
I 0
/ /
/
l
l
kr~ds'Z/~o/_J_.~o/~6/ 0
0
3.1
6 , 2 12 25 50 OCI~ ( p e r c e n t )
100
Fro. 2. Seedling injury response surfaces for oxygen concentration in the gas phase of the soaking solution (OCHG)-radiation dose interactions for "dry" barley seeds. nearly the same, the interactions with seed water content appear to be rather small. However, a close comparison of the radiation lines indicates that seeds of 4.1 and 6.1 (~'(~ water content react more alike at 5krad than do 1.8 and 4.1:'o water content seeds. At 15 and 20krad the radiation curves are very steep between 6.25 and 25°o O C H G on seeds of 1.8 and 4.1". water content, while on seeds of
6.1",, water content the slope is not as steep and continues to a higher O C H G . At 10krad seeds of 4.1°/0 water content showed a response more nearly centered between seeds of 1.8 and 6.1°/o/ water content. The seed water ContentO C H G interaction was the smallest of the double interactions and is easiest to see at the higher radiation doses and high O C H G , since seeds of 4.1 and 6.1 °/o water content responded
THE INTERACTION ON 7-IRRADIATED BARLEY SEEDS similarly, but seeds of 1.8'~, water content showed a tendency toward a q u a d r a t i c response. Between 6.25 and 12.5°,o O C H G seeds of 4.1°o water content responded more like seeds of 1.8~!/o water content. T h e surfaces themselves a m p l y demonstrate the great a m o u n t of interaction between O C H G and radiation dose. For each seed water content (Fig. 1 ), an arc can be d r a w n around the base of the start of this interaction. For example, the dotted line in Fig. 1C describes the arc for 6.1~Io seed water content in the lower water content experiment. As the seed water content is decreased, this arc moves toward a lower radiation dose with a lower O C H G . This same effect holds for seed water contents up to 10",o as is shown in Fig. 2. T h e response surfaces for O C H G - r a d i a t i o n exposure interaction for seeds of higher water content are shown in Fig. 2. T h e response due to seed water content was more nearly additive here than in Fig. 1. To compare these surfaces with those in the preceding figure, the u p p e r radiation level in this figure should be ignored. T h e injury on seeds of 6.1°~, water content resulting from 10 and 2 0 k r a d in this experiment was less than the injury ti~om similar treatment in the preceding experiment (Fig. 3). This difference must have been due to the difference in procedure used for the two experiments. T h e reduction in injury to these seeds would be due to cooling to - 7 8 ° C , or w a r m i n g back to 0~C. Neither should have 0 •
tO krads I0 krads; cooled t o - T B C
80
CI 20 kreds
70
159
caused a decrease in damage. ~st T h e change ill distance (° o injury) with a change in O C H G , between the lines for the respective radiation exposures, indicates a change in the m a g n i t u d e of the OE. T h e r e appears to be two components to the decrease in d a m a g e , one which effects the oxygen-independent d a m a g e and a second which influences the oxygend e p e n d e n t damage. T h e exact relationship could not be determined but there appears to be a small proportional decrease in d a m a g e with an increase in the radiation dose or in the m a g n i t u d e of the OE. T h e decrease was greater than would be expected from a strictlv additive response. T h e percentage of tile full oxygen enhancement (100~, o O C H G ) which was realized tbr each O C H G with each radiation exposure, summed over seed water content within each experiment, is shown in T a b l e 2. T h e values were calculated from the formula: Nitrogen S o a k i n g - - M i x e d Gas Soaking Nitrogen S o a k i n g I O x y g e n Soaking
x 100,
where each value was a seedling height in centimeters. In general, the higher the exposure of irradiation, the lower the O C H G must have been to show an oxygen enhancement or any portion of the full OE. A t 3.1°,o O C H G no consistent O E was obtained. An O C H G of 6.25°0 gave only a slight O E with the higher radiation exposures. Except for the low radiation exposures, about 25°o or more of the full O E was obtained with 12.5°~, O C H G , 5()~I~ or more with 2 5 ° ; O C H G , and 75%~, or more with 50~!o O C H G .
60 50 -
40
-~,
30
IO
O0
5I
6.25
02 Concentration
12 5 in t h e
25 SoaKing
50
o
I00
Gas (%)
FIG. 3. Effect of controlled (irradiated at 0°C and cooled to - 7 8 C) and uncontrolled temperature during irradiation and pri~w to soaking on the ~ccdlin~ i,,im> r,'Sl),ms," ,,l'l~,u'h'\ scotia. B
Radiosensitivity measured by a comparison of doses giving the same biological effect T h e radiation doses required to give 40°o injury to seeds at each of the five seed water contents soaked with each of the levels of O C H G are shown in Fig. 4. All of the O C H G curves indicate that there m a y have been a q u a d r a t i c c o m p o n e n t for seed water content. T h e q u a d r a t i c response for any one line m a y not be significant, but since this same response was obtained for the 7 soaking atmospheres,
160
E. DONALDSON, R. A. NILAN and C. F. KONZAK
Table 2. Percent oJ" the Jull oxygen enhancement obtained Jor each exposure of radiation used in barley seeds, summed over seed water content Oxygen conc. in the gas phase Seed water content 1",, )
Exposure (krad)
N 2 hyd* (cm)
5 10 15 20 10 20 30
11.51 11.34 10.55 10.47 12.68 12.57 11.84
2q5
6-10
3.1 6.25 12.5 25 50 Percent of full oxygen enhancement - 13.0 3.5 -2.7 0.7 3.8 5.6 1.4
1.5 12.2 2.6 10.0 2.5 10.2 14.0
23.6 36.7 26.6 43.7 19.4 25.9 38.0
34.2 62.5 52.3 68.6 41.3 46.5 70.1
79.4 84.5 81.5 90.0 73.5 74.5 90.4
Full Oz"~ enhancement (cm) 1.99 5.10 7.05 8.12 2.37 7.49 9.30
*Seedling height (cm) of the sample soaked in niu'ogen-bubblcd water. Average of 3 replications. ' F u l l oxygen enhancement as the difference het~een tile seedling height of the samples soaked in nitrogen and those soaked in 100t!i, oxygen.
~_~6
0
~
7
0 0.0%OCHG
50
625%0CHG
L~J
I0 ~
I~50CHG
C
H
G
SeedWaterContent(%) Fro. 4. Radiation doses required to give 40°0 injury to barley seeds of 5 water contents when soaked in each of 7 oxygen concentrations in the gas phase of the soaking solution (OCHG).
each line having been obtained from the average of three replications, the evidence was quite strong that, for the procedure used, the quadratic response did exist. T h e greatest change occurred between 2.0 and 4.3% seed water contents, with a more gra~tual change between seeds of 4.3 and 7.7°:o water content, followed
by a negative slope between seed water contents of 7.7 and 9.9~I0. The m a x i m u m change or reduction in radiosensitivity occurred between seeds of 2.0 and 7.7°:o water content and was greatest at 6.253o O C H G , being 14.7krad. T h e greater radiosensitivity of seeds of 9.9')o water content c o m p a r e d to seeds of 7.7~{~ water content was very consistent, ranging from a m a x i m u m of 4.Tkrad at 3.1°,b O C H G to 0 . 5 k r a d at 5 0 ° ' 0 0 C H G . At 100°o O C H G , seeds of 9.9".~, water content showed about the same level of radiosensitivity as seeds of 7.7~'o water content. A comparison of the 8.0 and 10.0°i, water content seed in graphs B and C in Fig. 2 shows that the 10.0°0 water content seeds have a greater radiosensitivity at intermediate O C H G than the 8.0°./0 water content seeds. T h e distance between the lines, Fig. 4, indicates the change in oxygen-dependent damage ( r a d i a t i o n - o x y g e n interaction) associated with the change in O C H G . Since the lines for 0.0 and 3.1~!{) O C H G and those for 25, 50 and l(/0°o O C H G were much closer together than those for the intermediate O C H G ' s , a sigmoid curve or cubic response was indicated. In Fig. 5 the base indicates an O E R of 1.0, as obtained with either a very low oxygen concentration or with a seed water content somewhat above 10 °':0" T h e area between the
THE INTERACTION ON 7-IRRADIATED BARLEY SEEDS
161
attempted to study different concentrations by mixing oxygenated water with oxygen-free water. With very dry seeds a mixture containing as little as 1°{~ oxygenated water gave 30 and 650o of the maximum oxygen effect with 10 and 20krad, respectively. One of the pro~5 blems with this system was its unsuitability for g determining the concentration of oxygen in the gas phase of the soaking solution (OCHG). For % Seed 2.~. instance, the actual dissolved oxygen conWater / centration for a mixture containing 1° o C°nfent6~ oxygenated water was not equivalent to 7.7j---"--'~7.7 1°~ O C H G but was 10.1°..o O C H G . Where the soaking water was continuously bubbled 0 3.1 6.25 12.5 25 ' ;o ,oo with various concentrations of oxygen in the gas 0 z Concentration in the Sooking Gas (%) FIG. 5. Oxygen enhancement ratio (OER) response phase, about 25, 50 and 80°o of the maximum surface fi)r oxygen concentration in the gas phase of oxygen enhancement (OE) was realized with respectively the soaking solution (OCHG)-barley seed water con- 12.5, 25 and 50c),,~ O C H G , (Table 2). Seed water content and radiation tent interactions. exposure both have an influence on the results. reaction surface and the expected surface (dot- The values published earlier (17) were slightly ted line) was the seed water c o n t e n t - O C H G higher than those reported here, but were averinteraction for this experiment. The area be- aged over radiation exposure rather than seed tween the base and the expected surface was water content and were for seeds of less than the O E for 10°~ water content seeds. This 6°o water content. The results from 'Fable 2 indicate that in the figure gives essentially the same information as " d r y " system the range of oxygen concentration Figs. 1 and 2, only in a different form. At the required to produce the O E during posthi~her seed water contents, namely 6.2, 7.7 radiation soaking is probably both broader and and 9.9(Io, the O E R values showed an apparent linear response from 6.25 to 100°'o O C H G when higher than the range required during irradiation in the "wet" system. An O C H G of over O C H G was plotted on a log scale. 3.1°.o is required for a consistent O E in irradiated barley seeds. The present study with DISCUSSION A N D CONCLUSIONS barley seeds does not establish an upper limit The purpose of varying the O C H G was to for the oxygen concentration influence. regulate the partial pressure of oxygen in the However, the need for an exponential increase atmosphere in equilibrium with the soaking in O C H G to obtain a linear increase in seedling water. The c~,mcntration of dissolved oxygen is injury indicates that a very large increase in the directly related to the partial pressure of oxygen dissolved oxygen concentration might be reif all other factors are held constant. The other quired to attain the level of injury realized with factors having the greatest influence on the irradiation of seeds in oxygen at high concentration of dissolved oxygen are tempera- pressures.(16) ture, dissolved salts or soluble organic comThe influence of seed water content on the pounds and barometric pressure. These would oxygen-dependent and independent damage ocnot have changed much during the course of an curring during soaking has been thoroughly reviewed {11'12) and will be discussed here only experiment, due to the methods used. In earlier reports, the effects of oxygen con- where relevant to this study. The radiosensicentration in the gas used for seed soaking was tivity of seeds soaked in oxygen-free water alexamined at three levels, 100, 20.9 (air) and tered little between seed water contents of 2.0 0",, Ils'~9'es~ using "'dry'" seeds. NIl.AN et al. t3"*) and 6.20~,, but decreased at 7.7°0 water (Fig. 3). a ._o
'
162
E. DONALI)SON, R. A. NIl,AN and C. F. KONZAK
This is in agreement with the results of CONOER et al. (~2) As the oxygen concentration increased
(logarithmically), radiosensitivity became almost linear at 1 2 . 5 ° / 0 0 C H G and remained linear with practically no change in magnitude up to 100% O C H G . The radiosensitivity followed the radical signal amplitude found by CONCER,(9) decreasing with increasing water content and was probably a simple reflection of this change in radical signal amplitude. The maximum injury was obtained at the lower seed water content, 2.0~I~,, which is similar to CONOER'S{8) results but differs slightly from those of CALDECOTT~6) and CONGER el al. ~x2) CALDECOTT(6) and CONGER el a/. ~12) obtained maximum seed radiosensitivity between 3 and 4°0 water content. Part of the difference in which seed water content showed the maximum radiosensitivity could be due to the method of presenting the results. The greatest interaction resulted from the difference between the i n j u r y response to O C H G of 1.8°o water content seeds and those of 4.1 and 6.10;0 seeds (Figs. 1, 2, 4 and 5). The influence is greatest at intermediate oxygen concentrations, which were not used in other investigations. The most striking effect of water content was greater radiosensitivity of the 10°o water content seeds. The reason for greater radiosensitivity of 10% water content seeds compared to 8 ', O water content seeds is not understood 'but could be due to the change in binding characteristics of water near the seed water content where the post-radiation OE ceases to occur. Seed water content interacts with oxygen concentration in at least two ways. As already noted, increasing the seed water content decreases the magnitude of the OE. ~x2'33) The other interaction involves the concentration of oxygen required to show an OE. As the seed water content is increased, the oxygen concenlration must also increase (Figs. 1 and 2) to obtain the same level of damage. As radiation exposure is decreased, the oxygen concentration must be increased to obtain the same level of damage. A triple interaction seems to occur. The interaction between radiation dose and oxygen concentration in the soaking gas is the most significant of the interactions studied in the present investigation. This interaction re-
flects the influence of oxygen concentration on the attainment of potential OE for the different exposures of radiation studied. Graphs of oxygen concentration vs percent injury provide a family of curves which have slopes increasing in steepness with an increase in oxygen concentration (Figs. 1, 2 and 5). A similar relationship is obtained by irradiating bacterial cells with high-intensity-pulsed electrons in the presence of increasing oxygen concentrations. (2°) In bacteria, the effect can be explained (14) by assuming that the first few krad of exposure deplete the oxygen within a cell by radiationinduced chemical reactions. The exposure required to deplete the oxygen by reaction would depend exclusively on the oxygen concentration. The rest of the exposure would be given in essentially anoxic conditions. The time for the soaking oxygen effect in seeds may be limited under certain conditions and the extent of reaction depends both on the oxygen concentration and the radiation exposure (16) as in the bacterial system. In the soaking oxygen enhancement, the level of effect depends on the oxygen diffused into the cells (of barley seeds), not the oxygen present at the time of irradiation. Since the magnitude of the reaction (Figs. 1, 2 and 5 show the completed reactions) is dependent on the radiation exposure and the time required for the elimination of oxygen sensitive sites (OSS), the oxygen-OSS reaction rate is dependent on OSS and oxygen concentrations, ~16) and interaction is the natural result. The decrease in damage due to the irradiation of seeds at 0°C and cooling to - 7 8 ° C (Fig. 3) was not expected and consequently was investigated only on seeds of 6°/o water content. The procedure was used as a convenience to prevent the decay of radicals in the more moist seeds during their transfer to the laboratory for soaking and to take advantage of the greater potential oxygen enhancement obtained from irradiating seeds at temperatures between 0 and 20°C as found by KRAtJSSE.~29) The reduction in damage due to immediate post-irradiation cooling of seeds is not the same as that resulting from the post-radiation heatshock used by KONZAK et a1328) and NIl.AN et a l / 3 e l The post-radiation heat-shock eliminated
THE INTERACTION ON ~,-IRRADIATED BARLEY SEEDS the soaking O E , gave a higher m u t a t i o n ti'equency with less chromosomal d a m a g e , and gave very little change in the seedling height. W h e n X - i r r a d i a t e d seeds were m a i n t a i n e d at 75-85°C for more than 4 8 h r , the d a m a g e decreased with an elimination of the oxygen enh a n c e m e n t ) 7) KESAVAN and KAMRA(24) found a similar response for heat-shock and microwaves. T h e elimination of the soaking oxygen enhancem e n t seems to be the major means of " r e d u c ing" d a m a g e to seeds in these experiments. T h e reduction in d a m a g e resulting from the i m m e d i a t e post-radiation cooling of seeds has its major influence on the o x y g e n - i n d e p e n d e n t damage. T h e effect of post-irradiation cooling on the o x y g e n - d e p e n d e n t d a m a g e is m i n i m a l and would p r o b a b l y be considered as part of the error, as BOTTINO(5) has done, if observed on only one point. T h e reduction in oxygend e p e n d e n t d a m a g e was greater for 2 0 k r a d than for 10krad and tended to be greatest for oxygen concentrations showing the most o x y g e n - d e p e n d e n t d a m a g e . CALDECOTT(7) obtained a similar reduction in the oxygend e p e n d e n t d a m a g e by heating seeds to 75°C for 2 5 h r prior to irradiation. H e a t i n g prior to irradiation could have a pre-conditioning effect on the possible sites for radicals, making them less receptive and resulting in less d a m a g e . This could not explain the post-radiation cooling effect. Both effects could be explained, however, by a change in the molecular structure accomp a n y i n g heat treatments or associated with a change in t e m p e r a t u r e , which would allow the harmless dissipation of a portion of the radiation energy absorbed. This change in molecular structure could be a shift in the three dimensional configuration of macromolecules moving reactive sites closer together or farther apart.
REFERENCES 1. AHNSTROM G. and MIKAELSEN K. (1968) Lifetimes of radiation-induced oxygen-sensitive centres in barley embryos soaked at different temperatures. Pages 283-286 in Mutations in plant breeding II, International Atomic Energy Agency, Vienna.
163
2. ALPER T. and MOORE J. L. (1967) The interdependence of oxygen enhancement ratios for 250 kVp X-rays and fast neutrons. Br. J. Radiol. 410, 843-848. 3. BAKER W. K. (1955) The oxygen effect and the mutation process. Brookhaven Symp. Biol. 8, 191200. 4. BOTCnEK C. M., KONZAKC. F. and NILAN R. A. (1964) Design of the 6°Co facility at Washington State University. IEEE Tram'. Prod. Eng. Prod.
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. CALDECOTT R. S. (1955') Effects of hydration oil X-ray sensitivity in Hordeum. Radiat. Res. 3~ 316 330. 7. CALDECOTTR. S. (1961) Seedling height, oxygen availability, storage and temperature: Their relation to radiation-induced genetic and seedling injury in barley. Pages 3-24 in Effects of ionizing radiation on seeds, International Atomic Energy Agency, Vienna. 8. CONGERA. 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, Publication 1355, Washington, D.C. 9. CONGERB. V. (1967} The influence of seed water content on the oxygen effect in irradiated barley seeds, Ph.D. Thesis, Washington State University. 91 p. 10. CONGER B. V., HILEMANJ. R., NILAN R. A. and KONZAK C. F. (1971)The influence of temperature on radiation-induced oxygen-dependent and -independent damage in barley seeds. Radiat. Res. 469 601~512. 11. CONGER B. V., NILAN R. A. and KONZAK C. F. (1968) Post-irradiation oxygen sensitivity of barley seeds varying slightly in water content. Radiat. Bot. 8~ 31 36. 12. CONGER B. V., NILAN R. A. and KONZAK C. F. (1969) The role of water content in the decay of radiation-induced oxygen-sensitive sites in barley seeds during post-irradiation hydration. Radiat. Res. 399 45 56. 13. CONGER B. V., NILAN 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. 69 129-144. 14. DEWEY D. L. and BOAG J. W. (1959) Modification of the oxygen effect when bacteria are given large pulses of radiation. .~ature (London) 183, 1450-1451.
164
1". I)()N,\IA)St)N. R . . \ . NIl,AN and (',. 1:. K()NZ.\K
15. DIxoN W. J. (1964) Analysis of variance, BMDO8V. In Biomedical computer programs, Health Services Computing Facility, University of California, Los Angeles. 16. DONALDSON E. (1970) The interaction of some factors influencing the oxygen effect in irradiated barley seeds. Ph.D. 'Thesis, Washington State University. 114 p. 17. DONALDSONE., .~ILAN R. A. and KONZAK C. F. (1968) The inlltlcnce of oxygen concentration on the post-irradiation oxygen effect in barley seeds. Barley Newsl. 11, 34-35. 18. EHRENBERGL. (1955) Studies on the mechanism of action of ionizing radiations in plant seeds. Sven. Kem. Tidskr. 67, 207-224. 19. EHRENBERG g. (1955) The radiation induced growth inhibition in seedlings. Bot. Not. 1118, 184215. 20. EPP E. R., WEiss H. and SANTOMASSOA. (1968) The oxygen effect in bacterial cells irradiated with high-intensity pulsed electrons. Radiat. Res. 3t, 320-325. 21. HARLEJ. R. (1963) Modification of radiobiological oxygen effects in very dry barley seeds. Ph.D. Thesis, Washington State University. 68 p. 22. HODGMEN C. D., ed. (1949) Handbook of chemistTy and physics. Chemical Rubber Publishing Co,, Cleveland. 23. HOWARD--FLANDERS P. and ALPER T. (1957) The sensitivity of micro-organisms to irradiation under controlled gas conditions. Radiat. Res. 7, 518-540. 24. KESAVANP. C. and KAMRAO. P. (1971) Studies on the postradiation modification of the gammaray induced damage in barley seeds by heatshock and microwaves. Pages 127 137 in R. A. NILAN, ed. Barley genetics II, Washington State University Press, Pullman. 25. KONZAKC. F., BOTTINOP. J., NILAN 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 see& II, International Atomic Energy A ffemx. Vienna. 26. KONZAK C. F., MIKAELSEN K., SIGURBJ6RN~SO:,; B. and BURTSCHERA. (1967) Recommended standard procedures for irradiating, cultivating and measuring cereal seeds to determine the effects of neutron irradiation in the neutron-seedirradiation programme. Pages 103-1(17 in Neutron irradiation of seeds, Technical Reports Series No. 76, International Atomic Energx Agency, Vienna.
27. KONZAK C. F., N1LAN R. A., HARLE J. R. and HEINER R. E. (1969) Control of factors affecting the response of plants to mutagens. Brookhaven Symp. Biol. 1t, 128-157. 28. KONZAKC. F., NILAN R. A., LECAVLTR. R. and HEINER R. E. (1961) Modification of induced genetic damage in seeds. Pages 155-169 in Effects of ionizing radiation on seeds, International Atomic Energy Agency, Vienna. 29. KRAUSSEG. W. (1968) Possibility of influencing radiosensitivity and mutability in barley. III. The combined effect of X-rays and various levels of temperature during irradiation and the influence of humidity and the stage of germination of the caryopses. Z. Pflanzenziichtg. 60, 19M,0. 30. LI C. C. (1964) Introduction to experimental statistics. McGraw-Hill, New York. 460 p. 31. MYHILL R. R. and KONZAKC. F. (1967) A new technique for culturing and measuring barley seedlings. Crop Sci. 7, 275 276. 32. NmAN R. A., 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. Pages 171-182 in Strahlenwirkung und milieu (Suppl. to Strahlentherapie, Vol. 51 ), Urban & Schwarzenberg, Munich. 33. NmAN R. A., KONZAK C. F., HARLE J. R. and LE~AtJLT R. R. (1963) The magnitude of the oxygen effect in irradiated barley seeds. Page 94 in S. J. GEERTS, ed. Genetics to@, Vol. 1, Pergamon Press, Oxford. 34. Nn,AN R. A., KONZAKC. F., LEGAUUrR. R. and HARLE J. R. (1961) The oxygen effect in barley seeds. Pages 139-154 in Effects of ionizing radiations on seeds, International Atomic Energy Agency, Vienna. 35. SPARRMANB., EHRENBERGL. and EHRENBERGA. (1959) Scavenging of free radicals and radiation protection by nitric oxide in plant seeds. Acta Chem. Scand. 13, 199-200. 36. STF.EL R. G. D. and TORRIE J. H. (1960) tSineiples and plocedures of statistics. McGraw-Hill, New York. 481 p. 37. THOMLINSON R. H. (1964) Treatment of animal tumors. Br. J. Radiol. 37, 720 (Abstract). 38. WASSERA. (1965) Beckma n Instructions 1223-C. Beckman Instruments, Inc., Fullerton, CA. 39. WITHERS R. and SCOTT O. C. A. (1964) 'The oxygen effect in normal tissue. Br. J. Radiol, 37, 720 (Abstract).