- Email: [email protected]
In vitro response of Dactylis glomerata leaf segments as affected by growing donor plants in radioactive soil
Envtronmenla/and Experimental BotaT9' , VoL. 33, No. 4, pp. 501--504, 1993
Printed in Great Britain.
00984472/93 $6.00 + 0.00 ~ 1993 Pergamon Press Ltd
IN VITRO R E S P O N S E OF D A C T Y L I S GLOMERA TA LEAF S E G M E N T S AS A F F E C T E D BY G R O W I N G D O N O R PLANTS IN R A D I O A C T I V E SOIL A. A. KONOPLYOVA,* L. V. ZSELTONOZSKAYA,* B. V. CONGER t and D. M. GRODZINSKY*~
*Department of Biophysics and Radiobiology, Institute of Cell Biology and Genetic Engineering, Kiev, Ukraine and ~'Department of Plant and Soil Science, The University of Tennessee, Knoxville, TN 37901-1071, U.S.A.
(Received 17 November 1992; in revisedform l 2 February 1993; acceptedin revisedform 13 May 1993) KONOPLYOVAA. A., ZSELTONOZSKAYAL. V., CONGERB. V. and GRODZINSKYD. M. In vitro reJponse of Dactylis glomerata leaf segments as affected by growing donor plants in radioactive soil. E N V I R O N M E N T A L AND E X P E R I M E N T A L B O T A N Y 3 3 , 5 0 1 - - 5 0 4 , 1993.--Sterile plants of Dactylis glomerata L. were grown for 2 months in sterile nonradioactive (control) and radioactive (treatment) soil. The radioactive soil was obtained from a 10-kin zone around the Chernobyl Nuclear Power Plant (Pripyat, Ukraine). After exposure, leaf segments were plated on to modified Murashige and Skoog medium containing 2.2 mg 1-l 2,4-dichlorophenoxyacetic acid. Somatic embryo formation and the average number of plantlets per explant were reduced 1.8 and 2 times, respectively, in the treated plants compared to the control.
Key words: Chronic irradiation, tissue culture, Dactylis glomerata L., orchardgrass, Gramineae, somatic embryo.
INTRODUCTION
ThE Nuclear Power Plant (NPP) accident at Chernobyl led to a wide area contaminated with a large n u m b e r of different radionuclides. The size of the disaster is well known: the a m o u n t of long-lived radionuclides released into the biosphere was 300-400-fold greater than that from the Hiroshima detonation. ~6/The physico-chemical form of the radionuclides resulting from the accident was unique. (9i T w o basic types of particles were released from the d a m a g e d reactor: large particles of dispersed fuel, and a wide range of sizes of wtrious types of particles on which
highly volatile components condensed. As a result, 9°Sr, 239pu and 24°pu (fuel particles) were deposited near the Chernobyl Plant, while 137Cs (condensation particles) was dispersed throughout Europe. ~2~ The unprecedented accident in Ukraine in 1986 was characterized by the simultaneous presence of 'classic' y-emitting radionuclides, condensed radionuclides (//-carriers) and 'hot' particles with c~-emitters in their matrix. ~l To investigate y-decay, it is necessary to explore ~and //-emitting radionuclides because of their high ionization density, great relative biological effectiveness, possible synergism of their reactions
++To whom all correspondence should be addressed. Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; MS medium, Murashige and Skoog medium. 501
A.A. KONOPLYOVA et al.
502
and problems with dosimetric measuring and apparatuses in the former U.S.S.R. In spite of the difficulties with estimation of the chronic irradiation dose rate in the 30-km zone of the Chernobyl NPP, it is important to know the ecological impact from the disaster in this region. Therefore, investigations of chronic irradiation effects on plants and animals are now underway. Various plant reactions can be observed from the influence of chronic irradiation in the immediate neighborhood of the Chernobyl NPP. Among these are various morphological abnormalities. The type and frequency of these abnormalities vary with radiation dose. Our study is an investigation of chronic irradiation effects due to radionuclide contamination of the soil on the response of Dactylis glomerata (orchardgrass or cocksfoot) leaf segments cultured in vitro. This species is a good candidate for our experiments. Orchardgrass is a typical plant growing in the 30-kin zone of the Chernobyl NPP. Also, techniques for in vitro leaf culture of this species are highly developed. (3'5i Somatic embryos arise in high numbers directly from mesophyll cells and scoring methods of embryo and plantlet formation are well described, iS,lO) Treatment of cell and tissue culture systems provides a potential for recovering mutants that are 'pure' or non-chimeric for a particular trait. Histological evidence suggests a single cell origin of embryos forming from orchardgrass leaf cultures. (1~i In addition, information already exists on the dose~response to y-radiation (6°Co) for development of somatic embryos and plantlet regeneration from Dactylis glomerata leaf segmentsJ 1°) MATERIALS AND M E T H O D S
Plant material Seeds of orchardgrass t~om a cross between an embryogenic genotype and a nonembryogenic genotype were surface sterilized for 1 rain in 70% ethanol, then for 10 rain in 0.1% HgCl2 and rinsed three times for 10 rain each in sterile distilled water. Sterile seeds ofDactylis glomerata were grown in sterile nonradioactive (NI, control) and radioactive (N2, treatment) soil. In order to
insure equivalent nutrient conditions during plant growth, 25 ml of sterile modified MS medium/8/ containing half quantities of macroelements, microelements and vitamins were supplemented in flasks with the soil twice a week. Flasks with the soil and seeds were incubated in natural summer light at 23 + 1°C in a greenhouse. In vitro cullure At the end of the 2 months the plants were harvested aseptically. The basal 15 mm of the two innermost leaves were used for culture in vitro according to methods previously described./3'5'm/ Culture was on a modified MS medium (s) containing 5 mg 1-1 thiamine, 1 g 1- l inositol, 3 g 1- l casein hydrolysate, 20/~M proline, 20/~M serine, 2.23 mg 1 J 2,4-D and solidified with 7 g 1-~ agar. Incubation was in the dark for 3 weeks at 25-t-I°C. After scoring for somatic embryo tormation, leaf segments with embryogenic callus were transferred to modified MS medium without 2,4-D and incubated for 3 weeks in the light at 23 + I°C. At the end of this period resulting plantlets were counted to obtain a quantitative estimate of embryo germination and plantlet development.
Sterilization of the .foil Flasks (250 ml) containing 80-90 g control and treatment soil were autoclaved twice (1.5 atm) tbr 40 min each with an interval of 1 week between autoclavings.
Radioactivity determinations Radioactivity of the soil was measured with a gamma-spectrometer based on an AM-A-02F1 analyser and a semiconductor (germanium) DGDK-80 detector. Total doses of external radiation for the vegetative growth period were estimated by thermo-luminescent dosimeters (DPG03, lithium-fluoride).
Statistical analysis Analysis of data was performed on all the above parameters with the Statistical Analysis System./j2i Standard errors were determined for each treatment.
DACTYL1S IN R A D I O A C T I V E SOIL
503
IN VITRO RESPONSE
Table l. Radioactivity of the soil for the growing of sterile Dactylis glomerata plants Experimental variant N1 control N2 treatment
Radionuclide
Radioactivity (Bq kg- ')
Counting uncertainty (%)
% of total content
Cs- 137 Cs- 137 Cs-134 Ce-144 Ru-106
30.00 1.239 x 10" 1.38l x 10~' 2.662 x 10:' 1.923 x 10~'
10.13 0.70 + 0.034 3.42_+0.31 8.19_+ 1 . 4 3 18.12_+ 1.40
100 67.58 + 2.74 7.53+0.47 14.47_+2.42 10.42_+ 1.52
N1, soil from outside the 30-km zone of Chernobyl; N2, soil from within the 10-km zone around Chernobyl.
RESULTS AND DISCUSSION
T h e total doses of external r a d i a t i o n o b t a i n e d by the control a n d t r e a t m e n t plants d u r i n g the 2m o n t h exposure were a p p r o x i m a t e l y 0.194 m G y a n d 0.105 Gy, respectively. D a t a on r a d i o a c t i v i t y o f the soil used tbr growing of the plants are shown in T a b l e 1. S o m a t i c embryos a p p e a r e d in the explants of the control and t r e a t m e n t regimens. T w o a n d three weeks after the initiation of culture, the n u m b e r of" explants with somatic e m b r y o s in the t r e a t m e n t g r o u p was reduced by 1.8 a n d 1.4 times, respectively (Fig. l). After scoring tor somatic e m b r y o tbrmation, leaf segments were transferred to M S
• 8 _rl
Irradiated Control
[
~ - 1 0 0 %
6 -
~59
-73%
m e d i u m without 2,4-D. T h e r e was a two-tbld decrease in the n u m b e r of plantlets regenerated from the tissue of chronically i r r a d i a t e d plants c o m p a r e d to that from n o n - i r r a d i a t e d plants (Fig.
2). W e previously r e p o r t e d that callus growth and p l a n t regeneration from explants of Nicoliana tabacum d o n o r plants growing inside a 10-km ' C h e r nobyl' zone were decreased due to the effects of" chronic i r r a d i a t i o n J 4) T h e results of that study are in a g r e e m e n t with our present d a t a on Dact~lis glomerata. T h e relative biological effectiveness of the chronic i r r a d i a t i o n conditions (external radiation plus internal r a d i a t i o n due to i n c o r p o r a t e d radionuclides) at C h e r n o b y l are a p p r o x i m a t e l y 10 times higher than the effectiveness of acute external 7-irradiation. <7! W e observed a two-fbld
• Irradiated 6 ~ 3 Con+.ro~ 5 --
-
-
- I00%
"6 03 Z
-2 I
--
"SzOeI ~
--
o --t~--- - -
2
3
Duration of culture (weeks)
FIG. l. Average number of explants with somatic embryo tbrlnation from Dactylis glomerata leaf segments cuhured in vitro tbr 2 or 3 weeks. The donors were previously grown tbr 2 months in nonradioactive soil (control') and radioactive soil (irradiated). Mean of 10 replications -+ S.E.
o LJ
I--E'-I -I°°°/°° 2
m:Y2
D u r a t i o n of
1-48% 3
culture (weeks)
FIG. 2. Average number of plantlets per explant from Dactylis glomerata leaf segments cultured in vitro for 2 or 3 weeks. The donor plants were previously grown for 2 months in nonradioactive soil (control) and in radioactive soil (irradiated). Mean of 10 replications ± S.E.
504
A.A. KONOPLYOVA et al.
reduction in average n u m b e r of plantlets when the total dose of external i r r a d i a t i o n of experimental plants for 2 months was 0.105 Gy, whereas the Ds0s for plantlet regeneration under the influence of external y-irradiation was 6.0 Gy. ~"~) It is concluded that exposure to low-level chronic radiation, including internal emitters, on orchardgrass plants very m u c h influenced the process of somatic embryogenesis in tissue culture. REFERENCES 1. BOROVOYA., BOGATOVS.~ KONOPLYOVA., STUKIN E., SUHORUCHKINA., PECHENKINS. and SCHETININ N. (1992) Phenomenology and methods of investigations of hot particles in 30-km zone Chernobyl NPP. Proceedings of the international symposium on radioecology: chemical speciation hot particles, Czechoslovakia, Znojmo, 12-16 October 1992, p. 100. 2. BORZILOVV. A. (1990) Source and transport processes inw)lved in contamination of the countryside following the Chernobyl accident (abstract), in Seminar on comparative assessment of the environmental impact of radionuclides released during three major nuclear accidents: Kyshtym, Windscale, Chernobyl, Luxembourg, 1 5 October 1990, p. 8. 3. CONGER B. V., HANNING G. E., GRAY D. J. and MCDANXEEJ. K. (1983) Direct embryogenesis from mesophyll cells of orchardgrass. Science 221, 850 851. 4. GROOZYNSKYD. M. and Loss A. A. (1990) Influence of chronic radiation on Nicotiana tabacum callusogenesis capacity in Abstr. 7 Int. Congress on Plant Tissue and (,'ell Culture, Amsterdam, 24-29June 1990.
5. HANNINGG. E. and CONGER B. V. (1986) Factors influencing somatic embryogenesis from cultured leaf segments of Dactflis glomerata. J. Plant Physiol. 123, 23 29. 6. KHITROVL. M. (1990) Chernobyl's ten lessons. In Seminar on comparative assessment of the environmental impact of radionuclides released during three major nuclear accidents: Kyshtym, Windscale, Chernobyl, Luxembourg, 1 5 October 1990, p. 6. 7. KUTLAI~MEOOVY. A., MmEEV A. N. and ZEZINA N. V. (1990) Non-classical radioecological phenomena in the biosphere after the Chernobyl accident. In Seminar on comparative assessment of the environmental impact of radionuclides released during three major nuclear accidents: Kyshtym, Windscale, Chernobyl, Luxembourg, 1 5 October 1990, p. 57. 8. MtJRASHmE T. and SKOOG F. (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 15, 473 497. 9. SOBOTOVmn E. V. (1990) Vertical migration of radionuclides ti~om the Chernobyl release in various landforms and geochemical conditions. In Seminar on comparative assessment of the environmental impact of radionuclides released during three major nuclear accidents: Kyshlym, Windscale, Chernobyl, Luxembourg, 1-5 October 1990, p. 32. 10. TOMASZEWSKIJR Z., CONGER B. V., BRUNNER H. and NOVAK F. J. (1988) Eflbcts of gamma radiation on the in vitro response of Dactylis glomerata leaf segments. Envir. Exp. Bot. 28, 335-341. 11. TRmIANO R. N., GRAY D. J., CONGER B. V. and McDANIEL J. K. (1989) Origin of direct somatic embryos tiom cultured leaf segments of Dactylis glomerata. Bot. Gaz. 150, 72 77. 12. [JRBAH V. Yu. (1964) Biometric methods [in Russian]. Nauka, Moscow.
Printed in Great Britain.
00984472/93 $6.00 + 0.00 ~ 1993 Pergamon Press Ltd
IN VITRO R E S P O N S E OF D A C T Y L I S GLOMERA TA LEAF S E G M E N T S AS A F F E C T E D BY G R O W I N G D O N O R PLANTS IN R A D I O A C T I V E SOIL A. A. KONOPLYOVA,* L. V. ZSELTONOZSKAYA,* B. V. CONGER t and D. M. GRODZINSKY*~
*Department of Biophysics and Radiobiology, Institute of Cell Biology and Genetic Engineering, Kiev, Ukraine and ~'Department of Plant and Soil Science, The University of Tennessee, Knoxville, TN 37901-1071, U.S.A.
(Received 17 November 1992; in revisedform l 2 February 1993; acceptedin revisedform 13 May 1993) KONOPLYOVAA. A., ZSELTONOZSKAYAL. V., CONGERB. V. and GRODZINSKYD. M. In vitro reJponse of Dactylis glomerata leaf segments as affected by growing donor plants in radioactive soil. E N V I R O N M E N T A L AND E X P E R I M E N T A L B O T A N Y 3 3 , 5 0 1 - - 5 0 4 , 1993.--Sterile plants of Dactylis glomerata L. were grown for 2 months in sterile nonradioactive (control) and radioactive (treatment) soil. The radioactive soil was obtained from a 10-kin zone around the Chernobyl Nuclear Power Plant (Pripyat, Ukraine). After exposure, leaf segments were plated on to modified Murashige and Skoog medium containing 2.2 mg 1-l 2,4-dichlorophenoxyacetic acid. Somatic embryo formation and the average number of plantlets per explant were reduced 1.8 and 2 times, respectively, in the treated plants compared to the control.
Key words: Chronic irradiation, tissue culture, Dactylis glomerata L., orchardgrass, Gramineae, somatic embryo.
INTRODUCTION
ThE Nuclear Power Plant (NPP) accident at Chernobyl led to a wide area contaminated with a large n u m b e r of different radionuclides. The size of the disaster is well known: the a m o u n t of long-lived radionuclides released into the biosphere was 300-400-fold greater than that from the Hiroshima detonation. ~6/The physico-chemical form of the radionuclides resulting from the accident was unique. (9i T w o basic types of particles were released from the d a m a g e d reactor: large particles of dispersed fuel, and a wide range of sizes of wtrious types of particles on which
highly volatile components condensed. As a result, 9°Sr, 239pu and 24°pu (fuel particles) were deposited near the Chernobyl Plant, while 137Cs (condensation particles) was dispersed throughout Europe. ~2~ The unprecedented accident in Ukraine in 1986 was characterized by the simultaneous presence of 'classic' y-emitting radionuclides, condensed radionuclides (//-carriers) and 'hot' particles with c~-emitters in their matrix. ~l To investigate y-decay, it is necessary to explore ~and //-emitting radionuclides because of their high ionization density, great relative biological effectiveness, possible synergism of their reactions
++To whom all correspondence should be addressed. Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; MS medium, Murashige and Skoog medium. 501
A.A. KONOPLYOVA et al.
502
and problems with dosimetric measuring and apparatuses in the former U.S.S.R. In spite of the difficulties with estimation of the chronic irradiation dose rate in the 30-km zone of the Chernobyl NPP, it is important to know the ecological impact from the disaster in this region. Therefore, investigations of chronic irradiation effects on plants and animals are now underway. Various plant reactions can be observed from the influence of chronic irradiation in the immediate neighborhood of the Chernobyl NPP. Among these are various morphological abnormalities. The type and frequency of these abnormalities vary with radiation dose. Our study is an investigation of chronic irradiation effects due to radionuclide contamination of the soil on the response of Dactylis glomerata (orchardgrass or cocksfoot) leaf segments cultured in vitro. This species is a good candidate for our experiments. Orchardgrass is a typical plant growing in the 30-kin zone of the Chernobyl NPP. Also, techniques for in vitro leaf culture of this species are highly developed. (3'5i Somatic embryos arise in high numbers directly from mesophyll cells and scoring methods of embryo and plantlet formation are well described, iS,lO) Treatment of cell and tissue culture systems provides a potential for recovering mutants that are 'pure' or non-chimeric for a particular trait. Histological evidence suggests a single cell origin of embryos forming from orchardgrass leaf cultures. (1~i In addition, information already exists on the dose~response to y-radiation (6°Co) for development of somatic embryos and plantlet regeneration from Dactylis glomerata leaf segmentsJ 1°) MATERIALS AND M E T H O D S
Plant material Seeds of orchardgrass t~om a cross between an embryogenic genotype and a nonembryogenic genotype were surface sterilized for 1 rain in 70% ethanol, then for 10 rain in 0.1% HgCl2 and rinsed three times for 10 rain each in sterile distilled water. Sterile seeds ofDactylis glomerata were grown in sterile nonradioactive (NI, control) and radioactive (N2, treatment) soil. In order to
insure equivalent nutrient conditions during plant growth, 25 ml of sterile modified MS medium/8/ containing half quantities of macroelements, microelements and vitamins were supplemented in flasks with the soil twice a week. Flasks with the soil and seeds were incubated in natural summer light at 23 + 1°C in a greenhouse. In vitro cullure At the end of the 2 months the plants were harvested aseptically. The basal 15 mm of the two innermost leaves were used for culture in vitro according to methods previously described./3'5'm/ Culture was on a modified MS medium (s) containing 5 mg 1-1 thiamine, 1 g 1- l inositol, 3 g 1- l casein hydrolysate, 20/~M proline, 20/~M serine, 2.23 mg 1 J 2,4-D and solidified with 7 g 1-~ agar. Incubation was in the dark for 3 weeks at 25-t-I°C. After scoring for somatic embryo tormation, leaf segments with embryogenic callus were transferred to modified MS medium without 2,4-D and incubated for 3 weeks in the light at 23 + I°C. At the end of this period resulting plantlets were counted to obtain a quantitative estimate of embryo germination and plantlet development.
Sterilization of the .foil Flasks (250 ml) containing 80-90 g control and treatment soil were autoclaved twice (1.5 atm) tbr 40 min each with an interval of 1 week between autoclavings.
Radioactivity determinations Radioactivity of the soil was measured with a gamma-spectrometer based on an AM-A-02F1 analyser and a semiconductor (germanium) DGDK-80 detector. Total doses of external radiation for the vegetative growth period were estimated by thermo-luminescent dosimeters (DPG03, lithium-fluoride).
Statistical analysis Analysis of data was performed on all the above parameters with the Statistical Analysis System./j2i Standard errors were determined for each treatment.
DACTYL1S IN R A D I O A C T I V E SOIL
503
IN VITRO RESPONSE
Table l. Radioactivity of the soil for the growing of sterile Dactylis glomerata plants Experimental variant N1 control N2 treatment
Radionuclide
Radioactivity (Bq kg- ')
Counting uncertainty (%)
% of total content
Cs- 137 Cs- 137 Cs-134 Ce-144 Ru-106
30.00 1.239 x 10" 1.38l x 10~' 2.662 x 10:' 1.923 x 10~'
10.13 0.70 + 0.034 3.42_+0.31 8.19_+ 1 . 4 3 18.12_+ 1.40
100 67.58 + 2.74 7.53+0.47 14.47_+2.42 10.42_+ 1.52
N1, soil from outside the 30-km zone of Chernobyl; N2, soil from within the 10-km zone around Chernobyl.
RESULTS AND DISCUSSION
T h e total doses of external r a d i a t i o n o b t a i n e d by the control a n d t r e a t m e n t plants d u r i n g the 2m o n t h exposure were a p p r o x i m a t e l y 0.194 m G y a n d 0.105 Gy, respectively. D a t a on r a d i o a c t i v i t y o f the soil used tbr growing of the plants are shown in T a b l e 1. S o m a t i c embryos a p p e a r e d in the explants of the control and t r e a t m e n t regimens. T w o a n d three weeks after the initiation of culture, the n u m b e r of" explants with somatic e m b r y o s in the t r e a t m e n t g r o u p was reduced by 1.8 a n d 1.4 times, respectively (Fig. l). After scoring tor somatic e m b r y o tbrmation, leaf segments were transferred to M S
• 8 _rl
Irradiated Control
[
~ - 1 0 0 %
6 -
~59
-73%
m e d i u m without 2,4-D. T h e r e was a two-tbld decrease in the n u m b e r of plantlets regenerated from the tissue of chronically i r r a d i a t e d plants c o m p a r e d to that from n o n - i r r a d i a t e d plants (Fig.
2). W e previously r e p o r t e d that callus growth and p l a n t regeneration from explants of Nicoliana tabacum d o n o r plants growing inside a 10-km ' C h e r nobyl' zone were decreased due to the effects of" chronic i r r a d i a t i o n J 4) T h e results of that study are in a g r e e m e n t with our present d a t a on Dact~lis glomerata. T h e relative biological effectiveness of the chronic i r r a d i a t i o n conditions (external radiation plus internal r a d i a t i o n due to i n c o r p o r a t e d radionuclides) at C h e r n o b y l are a p p r o x i m a t e l y 10 times higher than the effectiveness of acute external 7-irradiation. <7! W e observed a two-fbld
• Irradiated 6 ~ 3 Con+.ro~ 5 --
-
-
- I00%
"6 03 Z
-2 I
--
"SzOeI ~
--
o --t~--- - -
2
3
Duration of culture (weeks)
FIG. l. Average number of explants with somatic embryo tbrlnation from Dactylis glomerata leaf segments cuhured in vitro tbr 2 or 3 weeks. The donors were previously grown tbr 2 months in nonradioactive soil (control') and radioactive soil (irradiated). Mean of 10 replications -+ S.E.
o LJ
I--E'-I -I°°°/°° 2
m:Y2
D u r a t i o n of
1-48% 3
culture (weeks)
FIG. 2. Average number of plantlets per explant from Dactylis glomerata leaf segments cultured in vitro for 2 or 3 weeks. The donor plants were previously grown for 2 months in nonradioactive soil (control) and in radioactive soil (irradiated). Mean of 10 replications ± S.E.
504
A.A. KONOPLYOVA et al.
reduction in average n u m b e r of plantlets when the total dose of external i r r a d i a t i o n of experimental plants for 2 months was 0.105 Gy, whereas the Ds0s for plantlet regeneration under the influence of external y-irradiation was 6.0 Gy. ~"~) It is concluded that exposure to low-level chronic radiation, including internal emitters, on orchardgrass plants very m u c h influenced the process of somatic embryogenesis in tissue culture. REFERENCES 1. BOROVOYA., BOGATOVS.~ KONOPLYOVA., STUKIN E., SUHORUCHKINA., PECHENKINS. and SCHETININ N. (1992) Phenomenology and methods of investigations of hot particles in 30-km zone Chernobyl NPP. Proceedings of the international symposium on radioecology: chemical speciation hot particles, Czechoslovakia, Znojmo, 12-16 October 1992, p. 100. 2. BORZILOVV. A. (1990) Source and transport processes inw)lved in contamination of the countryside following the Chernobyl accident (abstract), in Seminar on comparative assessment of the environmental impact of radionuclides released during three major nuclear accidents: Kyshtym, Windscale, Chernobyl, Luxembourg, 1 5 October 1990, p. 8. 3. CONGER B. V., HANNING G. E., GRAY D. J. and MCDANXEEJ. K. (1983) Direct embryogenesis from mesophyll cells of orchardgrass. Science 221, 850 851. 4. GROOZYNSKYD. M. and Loss A. A. (1990) Influence of chronic radiation on Nicotiana tabacum callusogenesis capacity in Abstr. 7 Int. Congress on Plant Tissue and (,'ell Culture, Amsterdam, 24-29June 1990.
5. HANNINGG. E. and CONGER B. V. (1986) Factors influencing somatic embryogenesis from cultured leaf segments of Dactflis glomerata. J. Plant Physiol. 123, 23 29. 6. KHITROVL. M. (1990) Chernobyl's ten lessons. In Seminar on comparative assessment of the environmental impact of radionuclides released during three major nuclear accidents: Kyshtym, Windscale, Chernobyl, Luxembourg, 1 5 October 1990, p. 6. 7. KUTLAI~MEOOVY. A., MmEEV A. N. and ZEZINA N. V. (1990) Non-classical radioecological phenomena in the biosphere after the Chernobyl accident. In Seminar on comparative assessment of the environmental impact of radionuclides released during three major nuclear accidents: Kyshtym, Windscale, Chernobyl, Luxembourg, 1 5 October 1990, p. 57. 8. MtJRASHmE T. and SKOOG F. (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 15, 473 497. 9. SOBOTOVmn E. V. (1990) Vertical migration of radionuclides ti~om the Chernobyl release in various landforms and geochemical conditions. In Seminar on comparative assessment of the environmental impact of radionuclides released during three major nuclear accidents: Kyshlym, Windscale, Chernobyl, Luxembourg, 1-5 October 1990, p. 32. 10. TOMASZEWSKIJR Z., CONGER B. V., BRUNNER H. and NOVAK F. J. (1988) Eflbcts of gamma radiation on the in vitro response of Dactylis glomerata leaf segments. Envir. Exp. Bot. 28, 335-341. 11. TRmIANO R. N., GRAY D. J., CONGER B. V. and McDANIEL J. K. (1989) Origin of direct somatic embryos tiom cultured leaf segments of Dactylis glomerata. Bot. Gaz. 150, 72 77. 12. [JRBAH V. Yu. (1964) Biometric methods [in Russian]. Nauka, Moscow.