Influence of caffeine on the genetic effects induced by Ethyl Methanesulphonate (EMS) and on the recovery from the EMS-induced injury in barley

Mutation Research, 25 (1974) 3o5-3 Io

© Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

305

I N F L U E N C E OF C A F F E I N E ON T H E G E N E T I C EFFECTS I N D U C E D BY E T H Y L M E T H A N E S U L P H O N A T E (EMS) AND ON T H E RECOVERY FROM T H E EMS-INDUCED I N J U R Y IN B A R L E Y

T. GICHNER AND J. VELEMfNSK~" Institute of Experimental Botany, Flemingovo 2, z60 oo Prague 6 (Czechoslovakia)

(Received April 22nd, 1974)

SUMMARY

Barley seeds were treated with ethyl methanesulphonate (EMS) for 5 h, washed for 18 h, exposed for 3 h to caffeine, re-dried to 3o% water content and either immediately sown or stored fo~ IO days at 25 °. The caffeine post-treatment considerably potentiated the EMS-induced M1 germination and the Mx seedling height reduction, but had no influence on the EMS-induced M1 sterility or percentage of M, chlorophyll mutants. The caffeine post-treatment also had no significant influence on the "storage" recovery from the EMS-induced genetic effects.

INTRODUCTION

A vast number of papers deal with the influence of caffeine on the toxic and genetic effects induced by various kinds of mutagen in pro- and eukaryotic systems (for a list, see ref. I). In higher plants, caffeine is reported to potentiate the frequency of chromosome aberrations induced by various alkylatingn,l*,*°, 21 and non-alkylating mutagens n,12,18,29 and by gamma-rays~, 28. To our knowledge, no results dealing with the interaction of caffeine and mutagens on the induction of "gene" mutations in higher plants have yet been reported. One of the aims of the experiments described in this paper was therefore to study the possible effects of caffeine and EMS on the frequency of occurrence of recessive chlorophyll mutants in barley. A stimulus for these studies was that in other systems, as e.g., bacteria, Chinese hamster cells, etc., conflicting results are reported: caffeine potentiation~,13,~s,19, 2e as well as caffeine diminution*2,23, ~7 of the yield of mtrtations induced by, e.g., UV and alkylating agents. Furthermore, neither synergistic nor antagonistic effects of caffeine with various mutagens were found in the mouse dominant lethal tesO. The synergistic and antagonistic effects of caffeine on mutagen-induced genetic effects are mostly explained by the caffeine inhibition of various repair systems Abbreviation: EMS, ethyl methanesulphonate.

306

T. GICHNER, J. VELEMiNSK~"

(cf. refs. I4, 15, 17, 22, 23). In barley, a system has recently been developedT/~, 2',25 which enables the study of recovery and repair from mutagen-induced injury. This recovery, which includes M1 germination and Mt seedling height reduction, MI sterility, chromosome aberrations and M~ recessive chlorophyll mutations, is achieved by storing mutagen-treated seeds with 3o% water content and is accompanied bv the repair of single-strand breaks and/or alkali-labile sites in DNA. The second aim of the present study was thus to find out if this recovery system is sensitive to caffeine treatment. MATERIAL AND METHODS

Seeds of the hulled barley variety Dvoran (diameter 2.5 2.8 mm) were treated with water-dissolved EMS for 5 tl at 25 °, washed for 17 h at 25 ~, sterilized in o.i% Lastanox (Lachema, Brno) for 3o min and then washed for an additional 3o rain. Alter the sterilization and washing, the seeds were exposed to caffeine for 3 h at 25 ° and rinsed for 2 3 min in water. Seeds were then redried at 4 o~ to 3o% water content and either immediately sown or stored above sterile water in a desiccator at 25" for IO days. The EMS treatments considerably delayed the seed germination as compared with control treatments, so that caffeine was applied on non-germinating seeds. To ensure caffeine application at comparable stages of germination, the control seeds were pre-soaked for only IO h. The methods for analyzing the M1 and M2 characters have been described elsewhere", ~. For each treatment, 800 to I2OO M~ seeds were sown, except for the controls with 2oo seeds. The values of M1 fertility (determined on IOO plants) and M, chlorophyll mutants are means from 4 growing replications. The significance of differences (P = 0.05) were calculated according to the analysis of variance. RESULTS

Treatment by caffeine alone (up to 3o mM) on water-presoaked seeds only slightly decreased the M1 seedling height (Fig. I). Post-treatment by caffeine strongly potentiated the EMS action on M1 seedling height, e.g. after 14o m M EMS treatment the seedling height was reduced to 7o% ; post-treatment with 15 m M caffeine brought the seedling height down to 4% of the control. The results in Fig. I thus reveal a clear-cut caffeine potentiation effect of EMS-induced M1 seedling height reduction. Caffeine post-treatment also potentiated the effect of EMS on M1 seed germination (Table I). For instance, after 3o m M caffeine treatment alone, the M1 germination was 86%, and after 9 ° m M EMS treatment 82.6% ; that is, in both to the level of the control. Treatment of seeds with 9 ° m M EMS and post-treatment with 3o m M caffeine led to complete lethality. The degree of EMS potentiation was dependent on the caffeine concentration, as evidenced by the decreased M1 germination with increasing dose of caffeine post-treatment. However, the caffeine potentiation of EMS-induced M1 germination reduction was not accompanied b y a significant decrease in M1 fertility or by a significant change in the output of M~ chlorophyll mutants. In the range of 5 to 25 m M caffeine posttreatments, the fertility varied from 53.5 to 68.2% and the frequency of M2 mutants from 1.6 to 2.4~o.

I N F L U E N C E O F C A F F E I N E ON E M S - I N D U C E D

307

GENETIC EFFECTS

%

8O

i

460

;~0

60 mN EMS

90 mN 0

5

10

IS

20

25

30

EMS

m M caffeane

F i g . I. I n f l u e n c e of c a f f e i n e p o s t - t r e a t m e n t o n t h e e t h y l m e t h a n e s u l p h o n a t e (EMS) induced M 1 s e e d l i n g h e i g h t r e d u c t i o n ( % of c o n t r o l ) . T r e a t m e n t c o n d i t i o n s : 5 h E M S a t 2 5 ° ; w a s h i n g f o r I 8 h a t 2 5 ° ; 3 h c a f f e i n e a t 2 5 ° ; r e d r y i n g a t 4 °0 t o 3 0 % w a t e r c o n t e n t . TABLE

I

INFLUENCE

OF CAFFEINE

POST-TREATMENT

ON THE GENETIC

EFFECTS

INDUCED

BY ETHYL

METHANE-

SULPHONATE ( E M S ) IN BARLEY SEEDS T r e a t m e n t c o n d i t i o n s : 5 h E M S a t 2 5 ° ; w a s h i n g f o r 18 h a t 2 5 ° ; 3 h c a f f e i n e a t 25 ° a n d r e d r y i n g a t 4 °0 t o 3 0 % w a t e r c o n t e n t .

EMS (raM)

Caffeine (raM)

Ms Ma germination fertility (%) (%)

M2 plants scored

M2 chlorophyll mutants (%)

90 90 90 90 9° 90 90

3° 25 20 15 IO 5 o

o 11.2 32.7 32.5 67.6 63.5 82.6

64.6 68.2 56.5 57.o 62.4 53-5

641 2573 2280 3861 4695 4563

2.1 1.6 2.0 1.9 2. I 2.4

o o o

3° 25 o

86.o 92.0 86. 5

81.8 77.3 80.2

4146 2036 4231

o.I o o

S i g n i f i c a n t d i f f e r e n c e ( P ~ 0.05) f o r M 1 f e r t i l i t y , 9 . 0 % ;

and for M 2 chlorophyll mutants,

0.80,0 .

The results in Table I I also show a synergistic effect of EMS and caffeine, expressed by the decreased MI germination, and reveal the insensitivity of EMS-induced Ms sterility and M2-chlorophyll mutation frequency to caffeine post-treatment (cf. treatment with EMS and caffeine without seed storage). The "storage" recovery from EMS-induced genetic effects was also insensitive to caffeine post-treatment. For instance, treatments with 14o m M EMS and posttreatments with 15 or IO m M caffeine caused complete field lethality or strongly decreased the percentage of M I seed germination. Storage of these treated seeds at

308 TAI3LI~

T. G I C H N E R ,

J. VELEM/NSKS"

11

I N F L U E N C E OF CAFF!~2INE POST-TREATMENT ON THE GENETIC EFFECTS IN BARLI~2Y SEEDS TREATFI) \VITH ETHYL METHANESULPHONATE ( E ~ I S ) AND STORED FOR IO I)AYS Treatment c o n d i t i o n s : 5 h FSMS a t 25 ; w a s h i n g 4 o ' t o 3 o o'o w a t e r c o n t e n t a n d s t o r a g e ; i t 25 .

I£MS (m31)

Cajjeim' (raM)

Storage (days)

f o r t 8 h a t -'.5 ; 3 h c a f f e i n e a t 25 ; r c d r y i n g

311 germination (",>)

311 .h'rtility (%)

14 °

I5

0

I40 [4 ° I4O

10 o 15

0 o lo

58.3 50. I

9-3 75.4

J4 ° 14 °

1o o

To Io

57 o 00. o

03.3 6 4 . ,5

(} .52. 4 73.5 4%4 {)9.8 74.o

31.3 28. I 73.4 72.7 73.1

15

o

1 I0 i 1o 1 to 110 1lo

IO o i5 i0 o

{} o 10 io 1o

90 9o 9° 9o 90 (}o

15 lo o 15 1o

o o o lo lo

o

o o o o

15 o 15 o

l

1o

Significant

difference (P

M ,a

5 1 .a

plants scored

chlorophyll mutants ( .o)

5° 9389 487t 5496

7.7 I .o t.2 i -4

] [6 1 i03

5.4

0

19. 3

7123 5261 6302

0. 4 o.() 1.o i .2.0 ] .0 -' "4 0.8 0.9 o.0 o o.o2 o o.o I

56.5 57.0 53-5 66.7 77.7

1o

32. 5 67.{> 82.6 69.0 73-{} 75.(}

7(). 2

2280 386~ 4563 6004 7229 68~ 7

o o lo 1o

7S.5 86.5 82. 5 74.0

75,o 80.2 73.8 76.4

282I 4231 3854 2968

0.05)

at

f o r MI f e r t i l i t y , 8 . 2 % ;

and

f o r M.~ c h l o r o p h y l l

nmtants,

I.~%.

3o% water content for io days caused a marked improvement of the .M, germination. At lower concentrations of EMS, the Io-day-storage treatment brought about an improvement of M~ fertility and a decrease in the amount of Me mutants, regardless of whether or not a caffeine post-treatment was applied. I)ISCUSSION

The results reported demonstrate that under the treatment conditions used: caffeine post-treatment potentiates the EMS-induced M1 germination and M~ seedling height reduction, but is without influence on EMS-induced M1 sterility and percentage of M2 chlorophyll mutants; and (b) caffeine post-treatment does not inhibit the "storage" recovery from EMS-induced genetic effects. In higher plants, eaffzine potentiation has been reported for the action of various chromosome-breaking compounds in Vicia. When root tips were treated, only simultaneous or post-treatments with caffeine were effective. Front these and from autoradiographie studies, KmLMAN et al. u assumed that the potentiation of the yield of chromosome aberrations in Vicia is achieved only when the roots are exposed to caffeine during the S-phase. Experiments by ~ W I E T L I } N S K A et al. 2° have shown that caffeine pre-treatment on germinating seeds before DNA synthesis also potentiates the frequency of diepoxybutane-induced chromosome aberrations. In the experiments

(a)

INFLUENCE OF CAFFEINE ON E M S - I N D U C E D GENETIC EFFECTS

309

reported here, caffeine was applied on barley seeds 18 h after the EMS treatment. At this stage the mutagen-treated seeds had not yet entered semiconservative DNA synthesisS, 2s as indicated by the absence of seed germination. Two different explanations for these results can be considered. (a) A mechanism other than an S dependent one is responsible for caffeine potentiation. (b) Caffeine applied on non-synthetic stages persists in the seeds and first becomes active in the S-stage during seed germination. We can provide no satisfactory explanation as to why, in these experiments, caffeine neither potentiated the EMS-induced M1 sterility nor affected the percentage of M2 chlorophyll mutants. Recent results tend to reinforce the view that caffeine preferentially inhibits, in plant and animal cells, post-replication repairl°,11,16m. The "storage" recovery in barley, attained by storage of non-germinating seeds with 30% water content (i.e. without semi-conservative DNA synthesis in meristematic cells), is accompanied by the repair of single-strand breaks and/or alkali-labile sites in DNA during the storage period 2~,25. It is, therefore, probable that this "storage" recovery is achieved by the excision type of repair before DNA synthesis. If so, and if caffeine inhibits mostly the post-replication repair, our negative results with caffeine on "storage" recovery are quite comprehensible. The potentiation of EMS-induced M1 germination and M1 seedling height reduction by caffeine in those cases where seeds are sown immediately, and the lack of caffeine inhibition on "storage" recovery, could mean that in our system two types of repair are potentially active--one sensitive and one insensitive to caffeine. ACKNOWLEDGEMENTS

We are indebted to Prof. B. A. KIHLMAN (Uppsala), Prof. C. F. KONZAK (USA), Dr. J. j. ROBERTS (London), Prof. R. RIEGER (Gatersleben) and Dr. J. 2UK (Warsaw) for valuable discussion. REFERENCES

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