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Mutation Research, 71 (1980) 101--107 © Elsevier/North-Holland Biomedical Press

LIQUID-HOLDING-MEDIATED ENHANCEMENT OF THE FREQUENCY OF CHROMOSOMAL ABERRATIONS INDUCED BY ETHYLENEIMINE IN BARLEY EMBRYOS CULTIVATED IN VITRO

T. GICHNER, J. VELEMfNSK(/and M. ONDi~EJ

Institute of Experimental Botany, Flemingovo 2, 160 O0 Prague 6 (Czechoslovakia)

(Received 11 October 1979) (Revision received 10 January 1980) (Accepted 15 January 1980)

Summary

Barley embryos, deprived of endosperm, were treated with ethyleneimine (EI) and subjected to liquid holding in various media for 4--24 h. Liquid holding in media lacking a 1% sucrose provision led to manifold increase of the frequency of aberrant metaphases in root-tip meristems of EI-treated embryos, cultivated in vitro in a maximal medium after the Cessation of the liquid- holding period. The enhancement of the chromosomal damage was dependent on the duration and temperature of the liquid holding. Autoradiographic studies showed that no semi-conservative DNA replication took place under liquid-holding conditions leading to an enhancement of the frequency of aberrant metaphases in EI-treated embryos.

When UV-irradiated bacteria are kept in buffer before plating, a gradual increase occurs in the number of cells able to form colonies on a complex medium (Castellani et al., 1964; Ganesan and Smith, 1968; Ma~ek and Sedliakov~, 1977; Roberts and Aldous, 1949). This response has been called liquid-holding recovery and was explained as resulting from the extended period between the mutagenic treatment and the first post-treatment DNA replication which allows more time for the repair processes to proceed.

In yeast, however, two different responses to liquid holding were observed. In respiratory-competent cells treated with various mutagens, the liquid holding caused a decrease in lethality and conversion frequency, whereas in treated cells deficient in respiration the lethality and number of gene conversions were enhanced (Zimmermann, 1968). The results were interpreted by the absence of repair in respiratory<teficient cells owing to the lack of energy supply after the consumption of intracellular fermentable substrates.

The experiments to be described here were designed to find out whether

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liquid holding can influence the frequency of induced genetic changes in higher plants. Barley embryos, deprived of their energy supply - - endospe rm ' -were treated with ethyleneimine (EI) and held for up to 24 h at a partially anaerobic condition in various media with or without provision of carbohydrate. The liquid-holding effects were expressed by the frequency of induced aberrant metaphases in root-tip meristems of EI-treated embryos, which were cultivated in vitro in a maximal medium after the cessation of the liquid holding.

Material and methods

Hulled seeds of Ametyst barley were soaked for 18--20 h in a 2% solution of chloramin B at 25°C and rinsed with sterile water for 10 min. The embryos were extirpated from the endosperm, placed on dry blotting paper in petri dishes and stored at 4--6°C for 1--12 weeks before the mutagenic treatment.

Dry extirpated embryos were treated for 2 h at 25°C with 5 or 7.5 mM EI (Koch-Light Laboratories) dissolved in 0.05M citrate--phosphate buffer adjusted to pH 7. After the mutagenic treatment, the embryos were washed in distilled water for 45 min and sterilized by a 15-min immersion in a 2% solution of chloramine B. The treated and sterilized embryos were held for 4-- 24 h at 0--25°C in the following media: (1) maximal medium, containing sucrose, inorganic salts and casein hydrolysate (Miflin, 1969) diluted 1 : 1; (2) minimal medium, containing inorganic salts of Miflin's medium diluted 1 : 1; (3) 0.35% casein hydrolysate; (4) solutions of 0.05--1% sucrose in distilled water; (5) 0.05 M citrate--phosphate buffer adjusted to pH 7; (6) distilled water.

All manipulations with embryos (extirpation, mutagenic treatment, washing, sowing) were accomplished under sterile conditions. The citrate--phosphate buffer, the liquid-holding medium and the cultivation medium were sterilized twice at 100°C for 30 min with a 24-h interval.

For each treatment variant, 110 embryos were held in vials (2 cm diameter) containing 10 ml of the liquid-holding medium. After the liquid-holding period, the embryos were transferred to vials containing 4 ml of maximal medium. The vials with 11 embryos each were shaken on a rotary shaker (140 rpm) at 25°C.

Aberrant metaphases were scored in root tips of embryos cultivated in maximal medium for 36--96 h (i.e. fixation time) either before or after the liquid holding. The embryos were pre-treated with 0.05% colchicine for 2 h at 25°C fixed in ethanol : acetic acid (3 : 1). Squash preparations were stained by the Feulgen method. In metaphases, 4 types of aberration were scored: (1) breaks, (2) chromatid translocations, (3) dicentrics, (4) rings. The frequency of chromosomal aberrations in controls (3 h water-soaked embryos) that were exposed to and not exposed to the liquid-holding regimen varied from 1 to 5%.

To determine whether any DNA replication took place in the course of liquid holding in distilled water, the labelling index (LI) was estimated. EI-treated embryos were held for 4--24 h in [3HTdR at 1 gCi ml-' (23.7 mCi/ mmole) at 25°C. Cytological preparations were made after fixation of the liquid-held embryos and stained with Schiff's reagent. The separated roots, apices and third-leaf meristems were squashed on a slide and coated with Ilford 14 emulsion, exposed for 10 days at 5°C and developed and mounted in

1 0 3

Euparal. 10 root tips with at least 5 0 0 0 cells each were evaluated for each liquid-holding period. Labelled cells were identified by the presence of more than 3 grains in the nucleus. As a positive control, EI-treated or control embryos were held for 20 h in distilled water and cultivated for 48 h in maximal medium containing [3H]TdR at 1 pCi/ml-' . The autoradiographic preparations were made as described above.

Results

Treatment of barley embryos with 5 mM EI for 2 h resulted in a low frequency of aberrant metaphases which never exceeded 10.3% (Table 1). However, liquid holding of the EI-treated embryos for 24 h in citrate--phos- phate buffer (pH 7) led to an increase in the frequency of chromosomal aberra- tions to a value of 87%. (The frequency of aberrations is expressed by the highest value from 3 fixation times.) A similar enhancement occurred when the EI-treated embryos were held for 24 h in a minimal medium and in a distilled water solution of 0.35% casein hydrolysate. Only a 24-h liquid holding of EI- treated embryos in a maximal medium did not result in an increase in fre- quency of aberrations above that found in root tips of embryos that were not subject to liquid holding. The maximal medium contained 0.35% casein hydro- lysate, the same inorganic salts as are in the minimal medium and 1% sucrose. Since liquid holding of the EIotreated embryos in the first two solutions led to an increase of the induced genetic injury, it could be assumed that the 1%

T A B L E 1

E F F E C T O F L I Q U I D H O L D I N G O F E I - T R E A T E D B A R L E Y E M B R Y O S F O R 24 h IN C I T R A T E - - P H O S P H A T E B U F F E R ( p H 7), M I N I M A L M E D I U M , 0 . 3 5 % C A S E I N H Y D R O L Y S A T E O R M A X I M A L M E D I U M O N T H E F R E Q U E N C Y O F A B E R R A N T M E T A P H A S E S

Treatment condi t ions: ext irpated e m b r y o s were treated with 5 m M E1 (pH 7) f o r 2 h a t 2 5 ° C , w a s h e d 1 h at 25°C, held for 2 4 h in various media at 25°C and cult ivated in max/real m e d i u m at 2 5 ° C . B, b r e a k s ; T, chromat id translocat ions; D, dicentrics; R, rings,

Liquid Liquid F ixat ion Metaphases Aberrant hold ing holding t ime scored metaphases (h) m e d i u m (h) (%)

B T D + R

0 - - 42 3 0 0 5.0 13 2

0 - - 48 300 6 .0 15 3

0 - - 54 300 10.3 28 3

2 4 B u f f e r 6 6 2 0 0 7 8 . 5 2 5 7 18 5 8 2 4 p H 7 7 2 2 0 0 85 .0 4 7 3 1 0 8 4 2 4 7 8 2 0 0 8 7 . 0 4 8 3 3 6 9

2 4 Min ima l 6 6 2 0 0 7 6 . 5 2 7 3 14 51 2 4 m e d i u m 7 2 2 0 0 8 1 . 0 3 8 5 11 8 0 2 4 7 8 2 0 0 8 9 . 0 4 2 3 1 8 4

2 4 Case in 6 6 2 0 0 7 9 . 5 3 9 6 8 7 8 2 4 h y d r o l y s a t e 7 2 2 0 0 7 9 . 0 4 2 1 7 8 5 2 4 7 8 2 0 0 8 5 . 5 4 7 2 6 7 9

2 4 M a x i m a ! 6 0 3 0 0 1 0 . 3 26 7 1 2 4 m e d i u m 6 6 3 0 0 7 .7 23 2 - - 2 4 7 2 3 0 0 7 .8 1 9 3 - -

1 0 4

%

9o~

7O

U 9

i: . . . . . . . . . . . . . . . . . . - 6 . . . . . . . . . . . . .

I o.'s ' o . ~ ' o

g •

i o.'5 o.~ 'o

Fig. 1. F r e q u e n c y o f a b e r r a n t m e t a p h a s e s in r o o t - t i p m e r i s t e m s o f e x t i r p a t e d b a r l e y e m b r y o s t r e a t e d w i th EI a n d s u b j e c t e d t o l i q u i d h o l d i n g in va r i ous c o n c e n t r a t i o n s o f s u c r o s e . T r e a t m e n t c o n d i t i o n s : e m b r y o s t r e a t e d w i t h 7 .5 m M El (A) o r 5 m M EI (B) fo r 2 h a t 2 5 ° C , w a s h e d 1 h a t 2 5 ° C a n d he ld fo r 1 8 h (A) o r 2 4 h (B) in 0 - - 1 % suc rose s o l u t i o n s . T h e d a s h e d l lne r e p r e s e n t s t he f r e q u e n c y o f a b e r r a n t m e t a p h a s e s in r o o t - t i p m e r i s t e m s of E l - t r e a t e d e m b r y o s l a c k i n g a l i qu id h o l d i n g . F o r e a c h f i x a t i o n t i m e 2 0 0 m e t a p h a s e s were s c o r e d .

sucrose provision in the maximal medium was responsible for preventing the increase in the frequency of EI-induced aberrant metaphases.

Fig. 1 shows that the frequency of chromosomal aberrations induced by EI t reatment depended on the concentration of sucrose in the liquid-holding medium. If the medium contained 1% sucrose, i.e. the same amount as in maximal medium, the frequency of aberrations was only slightly above the fre-

%

B 7O

!i i " , i , i i

ID4,H~TK)N O f HOLOgI4G Ih) ~ T X J I E OF HOf, D ~ G ~ : )

Fig. 2. F r e q u e n c y o f a b e r r a n t m e t a p h a s e s in r o o t - t i p m e r l s t e m s o f e x t i r p a t e d b a r l e y e m b r y o s t r e a t e d w i t h EI a n d s u b j e c t e d t o l i qu id h o l d i n g f o r v a r i o u s d u r a t i o n s o r a t v a r i o u s t e m p e r a t u r e s . T r e a t m e n t c o n d i t i o n s : e m b r y o s t r e a t e d w i th 5 m M EI fo r 2 h a t 2 5 ° C , w a s h e d f o r I h a t 2 5 ° C a n d he ld in d i s t i l l ed w a t e r f o r 0 - - 2 4 h (A) o r f o r 2 4 h a t 0 - - 2 5 ° C . F o r e a c h f i x a t i o n t i m e 2 0 0 m e t a p h a s e s we re s c o r e d .

105

T A B L E 2

L A B E L L I N G I N D E X (LI) A N D N U M B E R O F G R A I N S P E R N U C L E U S IN R O O T M E R I S T E M S O F E M B R Y O S H E L D O R C U L T I V A T E D IN V I T R O IN M E D I A C O N T A I N I N G [ 3 H ] T d R

Nucle i s c o r e d LI G r a i n s / n u c l e i

A ~i :> 50 0 0 0 0 - - B 4 0 0 0 9 5 . 6 8 9 . 8 C 4 0 0 0 9 7 . 2 9 5 . 5

A. e x t i r p a t e d e m b r y o s t r e a t e d fo r 2 h w i t h 5 m M EI a n d h e l d f o r 4 . 8 , 12 , 16 a 20 . 2 4 h b in [ 3 H ] T d R a t 1 pCi m1-1 . F o r e a c h l i q u i d - h o l d i n g p e r i o d , 10 r o o t t ips we re e v a l u a t e d e a c h c o n t a i n i n g a t l eas t 5 0 0 0 cells, a 2 l abe l l ed n u c l e i ; b l abe l l ing e v a l u a t e d in r o o t , a p e x a n d t h i r d l e a f m e r l s t e m . B, e x t i r p a t e d embryos treated for 2 h w i t h 5 m M EI, he ld f o r 2 0 h in d is t i l led w a t e r a n d c u l t i v a t e d f o r 4 8 h in m a x i m a l medium c o n t a i n i n g [ 3 H ] T d R a t 1 ~Cl m1-1 . C, c o n t r o l ( u n t r e a t e d ) e m b r y o s , s a m e as B.

quency of aberrations in root tips of treated embryos that were not subject to liquid holding. With decreasing concentration of sucrose in the medium, the frequency of aberrations gradually increased, so that after liquid holding in a medium lacking sucrose (distilled water) about 90% of the metaphases scored were aberrant. The increase in frequency of aberrant metaphases was depen- dent on the sucrose content and took place both in embryos treated with 7.5 mM EI and held for 18 h (Fig. 1A) and in embryos treated with 5 mM EI and held for 24 h (Fig. 1B) in a liquid-holding medium. The increase in the frequency of aberrant metaphases in EI-treated barley embryos was dependent on the duration (Fig. 2A) as well as on the temperature (Fig. 2B) of liquid holding.

The labelling index is a measure of the percentage of nuclei in a meristem that were in the S phase or had entered the S phase during the exposure to [3H]TdR. Autoradiographic studies (Table 2) show that no DNA replication occurred in conditions leading to an increase in the amount of induced chromo- somal aberrations, i.e. liquid holding of the EI-treated embryos in a medium deprived of sucrose.

The 24-h liquid holding of treated and control embryos in media without or with a low sucrose provision reduced the number of germinated embryos as well as the length of roots compared with embryos that lacked the liquid holding. The decrease in germination and root length also took place after holding in maximal medium, but to a much lesser extent.

The increase in frequency of aberration after EI treatment and subsequent liquid holding of embryos, is mainly due to enhancement of breaks and cb_ro- mosome-type exchanges (dicentrics and rings) and to a lesser extent to chromatid exchanges. Among chromatid exchanges induced by EI treatment, there was a high frequency involving the centromere regions, as reported earlier (Nicoloff and Gecheff, 1976).

Discussion

The liquid holding of embryos in partial anaerobic conditions for up to 24 h prevents the germination of embryos and, as shown in Table 2, no DNA replica-

106

tion takes place. The liquid holding thus artificially prolongs the span between the mutagenic treatment and the first post-treatment replication as it does in bacteria and yeast. The delay of DNA replication allows more time either for the repair of induced DNA lesions (in conditions promoting repair} or for the accumulation of lesions responsible for genetic alternations (in the absence or slow-down of repair).

Furthermore, the liquid holding considerably reduces and finally brings to a standstill the aerobic respiration in embryos, which can, however, be replaced to a certain extent by other less efficient metabolic pathways, e.g. anaerobic glycolysis. In embryos deprived of the endosperms, as well as in respiratory- deficient strains of yeast (Zimmermann, 1968), the pools of fermentable carbo- hydrate reserves are rapidly depleted, so that the energy-requiring reactions in the cells, e.g. the repair processes, are inhibited. If carbohydrate is supplied to the holding media, the embryos as well as the yeast cells can partly utilize this source as an energy supply. The 1% sucrose provision in the holding medium seems to provide sufficient energy for the repair of the chromosomal lesions, thus preventing the enhancement of the frequency of chromosomal aberra- tions, whereas lower concentrations of sucrose (0.05--0.5%) prevent the increase of aberrations of sucrose to a lesser extent.

That the repair of DNA in embryos depends on the energy supply from the endosperm, if not supplied from the holding medium, has recently been demonstrated in barley (Velemfnsk~ et al., 1977). A 2-day liquid holding of seeds treated with N-methyl-N-nitrosourea in distilled water led to repair of the induced single-strand breaks, whereas the same liquid holding of extirpated embryos prevented this repair.

The following explanation of the enhancement of the frequency of aberrant metaphases due to liquid holding of EI-treated embryos can be considered. If EI-treated embryos are not subjected to liquid holding, only a fraction of the primary DNA lesions (alkylated sites) are converted via apurinic (apyrimidinic) sites to single-strand breaks. If liquid holding is intercalated between the muta- genic treatment and the first post-treatment DNA replications, the conversion of alkylated sites to single-strand breaks proceeds to a greater extent and, in the absence of repair, they accumulate. In addition, the explanation proposed by Zimmermann (1968) for the enhancement of gene conversion in respiratory<leficient yeast cells due to liquid holding can be considered: in the absence of repair the DNA lesions are more susceptible to the action of degradative enzymes, which can enlarge them, induce new ones or convert these lesions into irreversible (unrepairable) ones.

The former explanation, based on experiments with labelled alkylating mu- tagens and on DNA-sedimentation analysis in alkaline sucrose gradients (Veleminsk~ et al., 1973a,b) was postulated for the enhancement of the fre- quency of chromosomal aberrations, chlorophyll mutation etc., caused by 1--8 weeks storage {"solid holding"} of seeds treated with alkylating agents (Bender and Gaul, 1966; Gichner and Gaul, 1971; Gichner and Velemfnsk~, 1977; Konzak et al., 1964). The "solid holding" of the treated seeds with 15--20% water content (1) prevented seed germination, (2) prevented DNA replication (Gichner and Velemfnsk~,v1977), (3) considerably lowered the metabolic activity in the stored seed (Svachulov~ et al., 1973}, (4) inhibited DNA repair

107

and led to an accumulation of induced DNA single-strand breaks (Velemfnsk~ et al., .1973a). The first two events caused the prolongation of the time span between the mutagenic treatment and first post-treatment replication, which is the main presupposition for post-treatment modulation. The effect (e.g. inhibi- tion of repair) brought about by the 1--8 weeks "solid holding" were thus similar to the effect of a 24-h liquid holding in media lacking carbohydrate and led to similar genetic effects -- enhancement of the frequency of induced chro- mosomal aberrations.

References

Bender, K., and H. Gaul (1966) Nachw~/sche, Ri lcktrocknung und Lagerung bei AMS-behandelten Gersten- samen, Radiat. Bot., 6 ,505- -518 .

Castei/ani, A., J. Ja~ger and R.B. Setlow (1964) Overlap of photoreact lvat ion and liquid holding recovery in Escherichia coli B, Science, 143. 1170--1171.

Ganesan, A.K.0 and K.C. Smith (1968) Dark recovery processes in Escherichia coli irradiated with ultra- violet light, I. Effect of rec- mutants on l iquid holding recovery, J. Bacteriol., 96 ,365- -373 .

Gichner, T., and H. Gaul (1971) Storage effect following t rea tment of barley seeds with e thyl methane- sulfonate, I. Influence of seed moisture content , Radlat. Bot., 11, 53--58.

Gichner, T., and J. Velemfnsk~ (1977) Change of chromatid- to chromosome-type of aberrat ions by prolonging the G 1 cell phase after d ie thyl sulphate t reatment , Mutation Res., 45, 205--211.

Konzak, C. F., R.A. Nilan, E.E. Froese-Gertzen and R.J. Foster (1964) Factors affecting the biological act ion of mutagens, in: J. Velemfnsk~ and T. Glchner (Eds.), Induct ion of Mutations and the Mutat ion Process, Academia, Prague, pp. 123--132.

Ma~ek, F., and M. Sedliakov~ (1977) Pyr/midine dimer excision and DNA degradation during I/quid holding recovery in ultraviolet-irradiated Escherlchia col l K12 uvr + rec, J. Gen. Microblol., I 0 1 , 1 3 1 - - 133.

Miflin, B.J. (1969) A technique for sterile culture of germinating barley embryos, J. Exp. Bot., 20 (1969) 805--809.

Nicoloff, H., and K. Gecheff (1976) Methods of scoring induced chromosome structural changes in barley, Mutat ion Res., 34, 233--244.

Roberts , R.B., and E. Aldous (1949) Recovery from ultraviolet i rradiat ion in Escherichla coll, J. Bacteriol, 57 ,363- -375 .

Svachulovi , J., T. Gichner and J. Veleminsk~ (1973) Respirat ion in barley seeds treated with mutagenic methy l methanesulphonate and stored at different water contents, Biol. Plant., 15, 140--143.

Velemihsky, J., S. Zadra~li, V. Pokorn~, T. Gichner and J. ~vachulov~ (1973a) Storage effect in barley, Changes in the amount of DNA leslons induced by methy l and ethyl methanesulphonate , Mutat ion Res., 19, 73---81.

Velemfnsk~, J., S. Zadra~i/, V. Pokorn~, T. Gichner and J. Svachulov~ (1973b) Repair of singie-strand breaks and fate of N-7-methylguanine in DNA during the recovery from genetical damage induced by N-methyl-N-nitrosourea in barley seeds, Mutation Res., 17, 49--58.

Velemfnsky, J., S. Zadra~il, V. Pokorn~, and T. Giehner (1977) DNA repair synthesis s t imulated by muta- genie N-methyl-N-nitrosourea in barley seeds and free embryos, Mutat ion Rcs., 44, 43--51.

Zirnmermann, F.K. (1968) The effect of Hquid holding on chemical induced le thal i ty and mitot ic gone convers/on in Saccharomyces cerevlmi~, Mol. Gen. Genet., 103, 11--20.