Modification of barley seed radiosensitivity with microwave radiation—I. Effect of moisture content and post-radiation hydration

Radiation Botany, 1969, Vol. 9, pp. 443 to 448. Pergamon Press. Printed in Great Britain.

MODIFICATION OF BARLEY SEED RADIOSENSITIVITY WITH MICROWAVE RADIATION--I. EFFECT OF MOISTURE CONTENT AND POST-RADIATION HYDRATION OM P. KAMRA* aad P. C. KESAVAN Laboratory of Radiation Biology, Dalhonsie University, Halifax, N.S., Canada

(Received 2 Apr/l 1969) Abstract--Microwave radiation treatment of 50 sec duration at a continuous frequency of 2450 MHz and a power of 9.5 W restores post-radiation injury in dormant barley seeds. Microwave restoration of damage is maximum in dry seeds (3 per cent moisture) and negligible in moist seeds (11 per cent moisture). Data indicate that the component of damage restored by microwave radiation under the conditions of the experiment is the oxygen-dependent post-radiation effect. R~smn~--Un traitement d'une durde de 50 sec par des ondes courtes de frdquence continue 2450 MHz et d'une puissance de 9,5 W restaure le dommage se produisant apr6s irradiation dam les grains d'orge sees et en dormance. La restauration du dommage par les ondes courtes est maximale dam les grains sees (3% d'humidit6) et ndgligeable dans les grains hydratds (11% d'humitit6). Les donndes indiquent que la composante du dommage rdpar6 par les ondes courtes dam ces conditions expdrimentales est l'effet oxyg~ne-d6pendant apr~s irradiation. Zu~,,,,,~enfass,~ng--Straldenbehandlung mit Mikrowellen von 50 Sek. Dauer, einer kontinulerlichen Frequenz yon 2450 MHz und einer Energie yon 9,5 Watt heir die als Bestrahlungsfolge auftretenden Schgden in ruhenden Gerstensamen. Die Heilung der Sch~iden durch Mikrowellen ist bei trockenen Samen (3% Feuchtigkeit) maximal nnd kaum merklich bei feuchten Samen (11% Feuchtigkeit). Die Ergebnisse lassen darauf schliessen, dass die Komponente, deren Sch/idigung durch Mikrowellen-Bestraldung tinter den Versuchsbedingungen kompensiert wird, den sauerstofl'abh~ingigen, als Bestrahlungsfolge auftretenden Effekt darstellt. INTRODUCTION HEAT treatment of bacterial spores( T) and dormant barley seeds0,4,5,s.s) either before or after ionizing irradiation has been known to modify radiosensitivity though the mechanisms involved in thermorestoration or enhancement of radiation injury are unknown. I n dormant seed the maintenance of elevated temperature before irradiation leads to a reduction in radio-

sensitivity0,~,s) whereas post-radiation heat treatment of relatively long duration either enhances injury0,4) or leads to a reduction in damageA s) O n the other hand post-radiation heat treatment of a short duration, such as a 60 sec wet heat shock, reduces growth and chromosomal damage but increases seedling chlorophyll mutations. (S) The temperature of the biological material

*Present address: Consultant, Joint FAO/IAEA Division, International Atomic Energy Agency, Vienna, Austria. 443

444

OM P. KAMRA and P. C. KESAVAN

can be raised very quickly by irradiation in the microwave region of the electromagnetic spectrum. The present studies are an attempt to gain information on the effect of 12.25 cm microwave radiation on radiosensitivity of dormant barley seed of varying moisture content and its relation to the post-radiation oxygen effect.

moist filter paper in petri dishes at 21°+2°C for 8 days and the seed viability as well as seedling height were recorded as a measure of injury. Seeds producing a seedling less than 2.5 cm in height were considered as unviable. Shoot tips in all treatments were fixed in acetic alcohol (1:3) and cytologically examined for dicentric bridges and acentric fragments in the anaphase stage of the first mitotic division by the Feulgen squash technique.C4)

MATERIALS A N D M E T H O D S

Seeds of hull-less barley (Hordeum vulgare) var. Himalaya were stored in a desiccator over calcium chloride or no desiccant or 60 per cent glycerol and equilibrated to a moisture content of 3, 6 and 11 per cent respectively. The moisture content was determined in small aliquots of seeds, as the difference between the weight of seeds before and after drying in an oven for 48 hr at 100°C. For each treatment seventy-five seeds were sealed in three 2 ml glass ampoules (three replicates) in air, maintained at dry ice temperature (ca. --78°C) and exposed to a dose of 25 kR or 50 kR of gamma rays using a gamma cell (20000 Cie°Co) from Atomic Energy of Canada Limited at a dose rate of 144.1R/see. Ampoules with seed were placed in a cylindrical cavity with 7.6 cm diameter and 3.5 cm height and exposed to continuous 12.25 cm microwave radiation for 50 sec. The microwave power from a magnetron oscillator at 2450 M H z was fed into the treatment cavity by means of a coaxial cable with a magnetic coupling loop. Power was obtained from a probe inserted into a main waveguide carrying approximately 1 kw of power and the level adjusted by manipulating the probe penetration in the waveguide. Microwave power was measured by means of a coaxial directional coupler and a Hewlett Packard Type 431C power meter and maintained in the treatment cavity at 9.5 W + 5 per cent during the microwave treatment. Microwave and y-ray controls and entirely untreated controls were included in each experiment. Immediately after microwave posttreatment the ampoules were cut open and the seeds hydrated in either oxygenated water or oxygen-free water (degassed N2 bubbled) at 0 ° to 4°C for 24 hr. They were then grown on

RESULTS

As the moisture content increases, the amount of microwave energy absorbed by the seeds is expected to increase although the microwave exposure dose is kept constant within + 5 per cent. This was reflected in the viability of seeds exposed to microwave. The dry seeds were least damaged and the moist seeds (11 per cent most damaged (Table 1). Microwave post-treatment clearly restores gamma-ray induced damage as measured by the viability of the seeds and seedling height (Table 1) as well as chromosome aberrations (Table 2). This microwave effect is, however, greatly influenced by the moisture content of the seeds and the availability of oxygen during the postradiation hydration. Thus at 11 per cent seed moisture the microwave radiation seem incapable of restoring post-radiation oxygendependent gamma-ray induced damage whereas its efficiency in restoring radiation damage increases at lower seed moisture levels and is maximum in dry seeds (Table 1 and 2). Oxygen-dependent post-radiation damage is least exhibited by seeds of relatively high moisture content but is rather prominent in dry seeds.Ca) It seems clear from the data (Tables I and 2) that the 12.25 cm microwave post-treatment is able to restore oxygendependent post-radlation damage very effectively. The data also indicate that at least in dry seeds (3 per cent) microwave post-treatment may also restore post-radiation damage in the absence of oxygen during hydration (Table I). However, such a conclusion is not indicated at the level of chromosome damage at the initiation of mitosis in shoot tips (Table 2). It is to be

445

66 --H-H

.~

CXl

c'q~q

~-H

666 -H-flI-H

&&

6 -H 9r~D

o ~

.4.,;..,

OQ',~,,

+t~

666 --H-H'-H

b

%

~o_~

66 -H-H

6 -H

d~

m. 9 9

~9

.d

.~ o

66 -H-H

666

6 -H

II

9

66

Q ~

N i

II

~o u

& 0

zo2

dd

dd

c~cfc~ c~ 6

6 0

0

U

r~

446

.~ ~ &

66""

66

666

gg

~g

~g~

gg

gg

ooo

6

.e,

Z

~

] z~

~'~

~.~

u

c4

° ~

0

o

MODIFICATION OF BARLEY SEED RADIOSENSITIVITY noted that the kernels were not equilibrated to 3 per cent in the absence of On. Hence, there may be an oxygen-conditioned after-effect also when O~ free water is used for soaking. DISCUSSION

Ionizing radiations produce initially radiationinduced events that lead to observable biological effects (seedling injury, chromosome aberrations etc.). The initial radiation-induced events are physio-chemical in nature and at least some are apparently reversible in dry, slowly metabolizing dormant seeds. It is most probable that microwave post-treatment is restoring some of these reversible physio-chemical events thereby preventing their consequences in terms of observable biological effects. The nature of interactions between microwave radiation and radiation-induced reversible reactive species involved are not clear at present. Alteration of response to ionizing radiation injury by microwave treatment at 2800 M H z and an intensity of 100 mW/cm 2 has been reported by THOMSON et al.,O~,12) in dogs, rats and mice. Pretreatment of mice with microwaves reduce X-ray induced lethality01) while simultaneous exposure to microwave and sublethal X-irradiation modifies hematological response.0~) Post-radiation microwave restoration of injury reported in the present study was conducted at a microwave frequency of 2450 MHz and power level of 9.5 W. Microwaves in the region of 2800 M H z to 2450 M H z have a considerable moisture-dependent heating effect. This does not imply that there may be no non-thermal effects of microwaves. In fact non-thermal effects of microwave exposure have been recently reported on the behavioural pattern of birds at a frequency of 9300 MHzO0) and metabolism of bacterial cells at 13,600 MHz. (xs) The data presented here are at variance with the earlier reported work of KONZAK, NmAN and coworkers(4-6) who found a significant wet heat-shock restoration of radiation damage at 16 per cent seed moisture(4) with a maximum effect at 12 per cent moisture level(6) and a lower magnitude of effect at 4 per cent seed moisture. (5) In wet heat-shock (60°G for 90 see.) experiments the use of water as a medium for heat transfer

447

raises important questions concerning the connection of hydration with heat-shock effect. In microwave experiments no such complications arise. Microwave post-treatment produces significant restoration only in dry seed (3 per cent) and has very little effect at 6 per cent and no observable effect on seed with 11 per cent moisture content. Attempts at measuring the temperature of the seed during microwave exposure were abandoned because of (1) the effects of the electric and magnetic fields on the thermocouple and (2) the effect of the thermoconple probe on the uniform heating of the seeds. In order to measure the average temperature of the seed the thermocouple probe must be placed in the seed mass. However, the metal thermocouple probe causes gross distortion of the electric fields in the region and hence upsets the uniform heating of the seeds. On the other hand if the shielded thermocouple probe is placed just inside the cavity and not in the seed mass, then it effectively measures the temperature of the metal cavity which is not necessarily related to the temperature of the seeds. The whole problem of measuring the temperature of the seeds whether directly or by extrapolation is complicated by the fact that microwave heating takes place inside the seed. Since the thermal conductivity of the seed is very low, temperatures measured by any means on the outside of the seed could be expected to be in an error by an estimated 50 per cent. We have measured the temperature on the outside of seed mass in the ampoules at intervals after microwave exposure and the temperature was found to rise from - 10°F (as taken out of the dry ice) to about 110°F after a 50 sec exposure. But we have to bear in mind that this value is really lower than the true value ins/de the seed by a possible 50 per cent. Furthermore, measurements of the temperatures outside the seeds do not consistently reflect the inside temperatures which have an embryo-moisture dependent rise (Helpful discussions with Professor A. Cm~zLm~N and Mr. E. R~m of the Nova Scotia Technical College, Halifax, N.S., Canada, are gratefully acknowledged). In dry bacterial spores, POWERS and coworkersC~) have identified three main classes of

448

OM P. KAMRA and P. C. KESAVAN

effects that precede and lead to biological damage. A n entirely oxygen-dependent portion 'Class 1' ; and two oxygen-dependent components 'Class I I ' (an immediate effect) a n d 'Class I I I ' (occurring after irradiation, also termed as the 'free-radical component'). I n dry barley seed CONGER et al.(2) have found a new slowly developing storage effect, independent of oxygen and long-lived free radicals. I n barley seed critical experiments have not been conducted to show whether the oxygen-dependent d a m a g e can be subdivided into two classes such as POWERS' 'Class I I ' and 'Class I I I ' effects. Because oxygen was present during irradiation in the present study POWERS' 'Class I ' (oxygenindependent) effect cannot be separated from the oxygen-dependent 'Class I I and I I I ' effects. It is clear that at least 'Class I I I ' (postradiation oxygen-dependent) d a m a g e is restored effectively by microwave radiation. This is consistent with a lack of microwave effect on seeds with 11 per cent moisture which should show negligible 'Class I I I ' effects.(Sl Parallel studies with electron paramagnetic resonance spectroscopy on long-lived free radicals should give similar relationship since 'Class I I I ' d a m a g e is also the 'free-radical component'.

Acknowledgements--Financial support by the National Research Council of Canada and the Research Development Fund, Faculty of Graduate Studies, Dalhousie University is gratefully acknowledged. The authors wish to express their sincere appreciation to Professor A. CREELMANof the Nova Scotia Technical College, Halifax for his unfailing enthusiasm and help in effecting the microwave treatments. Grateful acknowledgement is made to Miss L. Sea,ARE for technical assistance.

REFERENCES 1. BEROBUSeHV. L. and CALDEeOTTR. S. (1963) The effects of preirradiation and post irradiation temperature treatments on the X-ray sensitivity ofseeds of Hordeum. Radiation Res. 20, 207-220.

2. CONGERB. V., NILAN R. A. and KONZAK C. F. (1968) Radiobiological damage: A new class identified in barley seeds stored after irradiation. Science 162, 1142-1143. 3. CONGERB. V., NILAN R. A. and KONZAK C. F. (1968) Post-irradiation oxygen sensitivity of barley seeds varying slightly in water content. Radiation Botany 8, 31-36. 4. KONZAK C. F., CURTIS H. J., DELIHAS N. and NILAN R. A. (1960) Modification of radiationinduced damage in barley seeds by thermal energy. Can.07. Genet. Cytol. 2, 129-144. 5. KONZAKC. F., NILANR. A., L~OAULTR. R. and I-IEINER R. E. (t961) Modification of induced genetic damage in seeds, pp. 155-169. In The Effects of Ionizing Radiations on Seeds. I.A.E.A. Vienna, Austria. 6. NILAN R. A., KONZAK C. F., WAGNERJ. and LEGAULTR. R. (1964) Effectiveness and efficiency of radiations for inducing genetic and cytogenetic changes. Radiation Botany Suppl. 5, 71-89. 7. POWERSE. L. (1961) Reversibility of K-irradiation-induced effect in dry biological systems. 07. Cellular Comp. Physiol. Supplement 1 58, 13-26. 8. SANTOS L S. (1964) Reduction of sensitivity to e°Co gamma rays in Phaseolus aureus Roxb through pre- or post-irradlation heat treatment of the seed. Radiation Botany Suppl. 5, 263-271. 9. SMITHL. and CALDECOTTR. S. (1948) Modification of X-ray effects on barley seeds by pre- and post-treatment with heat. 07. Heredity. 39, 173-176. 10. T~NER J. A., ROMERo-SmRgA C. and DAWE S. J. (1967) Non-thermal effects of microwave radiation on birds. Nature 216, 1139. 11. THOMSON R. A. E., MICHAELSONS. M. and HOWLAND J. W. (1965) Modification of Xirradiation lethality in mice by microwaves (Radar). Radiation Res. 24, 631-635. 12. THOMSON R. A. E., MICHAELSONS. M. and HOWLANDJ. W. (1967) Microwave radiation and its effects on response to X-radiatlon. Aerospace Med. 38, 252-255. 13. WBBB S. J. (1968) Inhibition of bacterial cell growth by 136 gc microwaves. .Nature 2111, 374--375.