Theoretical and Applied Genetics 41, 18-- 20 (1971) | b y Springer-Verlag 1971
The Effect of Gibberellic Acid on Chromosomal Aberrations in EMS and MMS Treated Pisum sativum Linn. V. G. NARSINGHANI and SUDHIR KUMAR D e p a r t m e n t of Genetics a n d P l a n t Breeding, A g r i c u l t u r a l E x p e r i m e n t S t a t i o n , U n i v e r s i t y of U d a i p u r (India)
Summary. The present experiments revealed a reduction in seedling height and damage at chromosomal level in P i s u m treated with E M S and M M S . F u r t h e r it was observed t h a t GA 3 post-mutagen treatment could reduce this damage considerably b y reducing the number of aberrations as well as chromosomal translocations. The GA 3 t r e a t m e n t also
increased seedling height as well as pollen fertility.
I t is k n o w n t h a t d u p l i c a t i o n s as well as t r a n s l o c a t i o n s do p r o v i d e an i m p o r t a n t source of g e n e t i c v a r i a b i l i t y which m a y y i e l d p l a n t s of useful t y p e s a n d w h i c h could be used f u r t h e r for b r e e d i n g of d e s i r a b l e plants.
For meiotic studies, the floral buds were fixed in Carnoy's fixative (6 parts ethyl alcohol, 3 parts chloroform and t p a r t acetic acid) and squashed in 1% aceto carmine stain.
A large n u m b e r of r e c i p r o c a l t r a n s l o c a t i o n s h a s been i n d u c e d b y v a r i o u s m u t a g e n s . R e c i p r o c a l t r a n s l o c a t i o n s are b y far t h e m o s t c o m m o n s t r u c t u r a l r e a r r a n g e m e n t f o u n d a n d are e a s i l y t r a n s f e r r e d to l a t e r generations. T h e y are n o t o n l y of t h e o r e t i c a l i n t e r e s t b u t are also of p r a c t i c a l a p p l i c a t i o n . H a g b e r g (1964) a n d K o n z a k et al. (t964) h a v e i n d i c a t e d t h e possibilities of usefully e m p l o y i n g reciprocal t r a n s l o c a t i o n s in b r e e d i n g p r o g r a m m e s . L a m m a n d Miravalle (1959) h a v e d e s c r i b e d a t e s t e r set of t r a n s l o c a t i o n s in _Pisum.
T h e d a t a p r e s e n t e d in T a b l e 1 show t h a t t h e r e was a r e d u c t i o n in seedling h e i g h t in E M S - a n d M M S t r e a t e d plants. H o w e v e r , this d a m a g e w a s n e u t r a l i sed w h e n t h e seeds were given p o s t - m u t a g e n t r e a t m e n t of 1000 p p m G A 3. T h e effect of G A 3 was m u c h m o r e e v i d e n t in E M S - t r e a t e d m a t e r i a l t h a n in the M M S . I n E M S - t r e a t e d m a t e r i a l t h e increase in h e i g h t was q u i t e significant a n d even s u r p a s s e d t h e controldby a b o u t 25 p e r cent. T h e increase in h e i g h t in M M S t r e a t m e n t was m o r e or less e q u a l to t h a t of t h e control. G a u r a n d N o t a n i (t960) using maize, H a b e r a n d L u i p p o l d (1960) w i t h w h e a t a n d M a t h u r (196t) w i t h p o t a t o h a v e r e p o r t e d t h e m o d i f i c a t i o n of p h y s i o logical d a m a g e b y m u t a g e n s t h r o u g h p o s t - t r e a t m e n t w i t h gibberellic acid. K u m a r (1967) has r e p o r t e d t h a t G A 3 p o s t - m u t a g e n t r e a t m e n t could r e d u c e t h e c y t o l o g i c a l d a m a g e in g a m m a - i r r a d i a t e d barley 9
T h e p r e s e n t e x p e r i m e n t s were u n d e r t a k e n to induce d u p l i c a t i o n s a n d t r a n s l o c a t i o n s in P i s u m using E M S a n d M M S as well as to see if gibberellic acid (GAs) t r e a t m e n t h a d a n y effect in m o d i f y i n g prod u c t i o n of these d u p l i c a t i o n s a n d t r a n s l o c a t i o n s .
Results and D i s c u s s i o n
Material and Methods Pea seeds of the variety 'Bounville' were presoaked in water for two hours and then treated with one dose each of E M S (0.25%) and M M S (0.0250) for ten hours. The solutions of mutagens were prepared in phosphate buffer adjusted to p H 7.0. At the end of the t0-hour period, the seeds were washed in distilled water for 10 minutes and resoaked in a 1000 p p m solution of gibberellic acid (GAa) for 8 hours. At the end of the t r e a t m e n t the seeds were again washed with distilled water. Four replications, each of 25 treated seeds were sown in wooden flats for recording seedling height. Another set of 100 seeds was sown in the field in 4 plots. The seedling height was recorded after 10 days. For mitotic studies, 25 treated seeds were placed in a petri dish lined with wet filter paper and the root tips were collected and fixed from the germinating seeds. The slides for mitotic studies were prepared b y hydrolysing the root tips with a mixture of 9 parts of 2% aceto orcein and I p a r t of N H C L and then staining with 1 ~o aceto orcein.
Table 1. Seedling injury and milotic studies Mutagenic treatment Control
Mitotic studies x *Seediing height (cms.) bridgesrods dots
% aberrations per cell
5.16 3.40
Nil 49
Nil 14
Nil 27
0.00 0.45
. E M S 0 . 2 5 % + 6.80
35
17
7
0.30
48
25
33
0.53
46
4
32
o.41
E M S 0.25%
GA t o00 p p m M M S 0.025% 3.00 MMS
o.o25~/ +
5.0o
GA
* Seedling height as recorded after 10 days x No. of cells scored -- 200
E f f e c t of Gibberellic Acid on C h r o m o s o l n a l A b e r r a t i o n s in Pisum sativum
Vol. 41, No. 1
19
T a b l e 2. Meiotic studies and pollen [ertility
No. of cells analysed
Treatment
E M S 0.25 % 260 E M S 0.25% + 232 GA t 000 p p m M M S 0.25% 278 M M S 0.025% + GA t96
Metaphase anomalies
Anaphase and teloTotal phase anomalies like -aberrant like rings, chains, dots bridges, laggards, dots, cells fragments, etc. rods, fragments, etc.
% aberration % age fertile per cell pollen grain
46
25
71
0.27
19
27
46
0.20
73
42 22
41 17
83 39
0.30 0.20
62 74
67
T a b l e 3- Metaphase I and I I meiotic anomalies Rings Treatment
Chains
No. of Rings I~ings cells of 3 of 4 analysed chro- chromoso- mosomes rues
E M S 0.25% 260 E M S 0 . 2 5 % + 232 GA 1000 p p m M M S 0.025% 278 MMS0.025% +196 GA 1000 p p m
Rings of 6 Total No. chro- offings mosomes
Chain of 2 chromosomes II
17
Chain of 3 chromosorues
Chain of 4 Total no. chromoso- of chains, rues
FragTotal No. of o 9 ,ments, cells showing 70 aDerranon dots, anomalies per ceu rods, etc.
t5
14
46
0.18
3
14
19
0.08
5
9
3
l
3
l
1
--
2
1
2
--
l0
7
--
17
7
3
1
11
14
42
0.15
1
1
1
3
2
--
1
3
16
22
0.11
T a b l e 4. Anaphase I and I I meiotic anomalies
Treatment
No. analysed of cells
Bridges
Laggards, dots, etc. rods, Total
%age aberrations per cell
E M S 0.25%
260
23
2
25
0.09
E M S 0.25% + GA t000 p p m M M S 0.025% MMS0.025% +GA
232
19
8
27
0.12
278 196
35 14
6 3
41 17
0,15 O.O9
The mitotic studies (Table 1) show that GA a can effectively modify the cytological damage caused by chemical mutagens in the same way as it does with physical mutagens (Kumar, 1967). However, the response to GA a treatment was variable. A marked reduction in the number of bridges and dots was observed in E M S + GAa-treated material. The number of rods was reduced after treatment with M M S + GA 3 but not after treatment with E M S + GA 3. In the meiotic studies (Tables 2, 3 and 4), GAa had more effect on the metaphase than on the anaphase in E M S - t r e a t e d material. The rings and chains were reduced whereas there was no effect on dots and rods. These results corroborate the evidence obtained in the mitotic studies. In M M S - t r e a t e d material, GA 3 treatment had a marked effect both at the metaphase and anaphase stages, the aberrations per cell decreasing considerably at both stages.
From these results it is evident that GA 3 is effective in reducing chromosomal damage, but at the moment it is difficult to indicate how this comes about. Varner (1964) has shown that gibberellic acid controls the synthesis of 0~ amylase in barley endosperm, and that it induces the synthesis of RNA which in turn helps to repair the cytological damage caused by physical and chemical mutagens. Gibberellic acid probably has something to do with the changed structural arrangement of the DNA molecules (after treatment with E M S and M M S ) . Alternatively, increased protein synthesis stimulated by GA a may be helping in the repair mechanism as suggested by Varner
(1964). From the results presented here, it appears that post-mutagen treatment with gibberellic acid could be used with advantage in reducing chromosomal aberrations. Gibberellic acid should be used only if one is interested in reducing the chromosomal aber-
20
V.G. Narsinghani and Sudhir K u m a r : Effect of Gibberellic Acid on ChromosomalAberrations in Pisurn salivurn
r a t i o n s with a view to o b t a i n i n g a n increased n u m b e r of m u t a t i o n s ; it should n o t be used if one is i n t e r e s t e d in obtaining duplications and translocations. T h e d u p l i c a t i o n s a n d t r a n s l o c a t i o n s o b t a i n e d are u n d e r g o i n g f u r t h e r o b s e r v a t i o n s to d e t e r m i n e which chromosomes are i n v o l v e d .
Acknowledgements The authors are grateful to Dr. H. N. Mehrotra, Prolessor and Head of the Department and to Dr. B. K. Srivastava, Director, for the interest taken and facilities provided. The senior author is grateful to the Department of Atomic Energy, Government of India, for the award of a Fellowship as well as the funds for carrying out this project.
Received November 25, 1969 Communicated by H. Stubbe
References 1. Gaur, B . K . , Notani, N . K . : Effect of Gibberellic Acid on barley. Rad. Res. 3, 339--350 (1960). -- 2. Haber, A . H . , Luippold, H . J . : Effects of gibberellin on gamma irradiated wheat. Amer. J. Bot. 47, 140--144 (t960). -- 3. Hagberg, A. : Use of induced translocations in directed production of duplications. Suppl. Rad. Bot. 5, 7 4 1 - 752 (t964). -- 4. Kumar, Sudhir: Effect of Gibberellic Acid o n gamma irradiated barley seeds. Ind. J. Gen. 27, t 54-- 156 (t 967). -- 5. Lamm, R., Miravalle, R. J. : A translocation tester set in Pisum. Hereditas 45, 417 to 440 (1959). -- 6. Mathur, P. B.: Reversal of gamma ray induced dormancy of potato tubers of Gibberellic Acid. Nature, Lond., 19o, 547--548 (1961). -- Nilan, R.A., Konzak, C.F., Wanger, J., Legault, R . R . : The use of induced mutations in Plant Breeding. Supplement to Rad. Bot. 5, 71--89 (1964). -- 8. Varner, J . E . : Gibberellic Acid controlled synthesis of L-amylase in barley endosperm. Plant Physiol. 39, 413-- 415 (1964).
Dr. Sudhir K u m a r Geneticist Department of Genetics and P l a n t Breeding Agricultural Experiment Station University of Udaipur Udaipur, Rajasthan State (India)
The Effect of Gibberellic Acid on Chromosomal Aberrations in EMS and MMS Treated Pisum sativum Linn. V. G. NARSINGHANI and SUDHIR KUMAR D e p a r t m e n t of Genetics a n d P l a n t Breeding, A g r i c u l t u r a l E x p e r i m e n t S t a t i o n , U n i v e r s i t y of U d a i p u r (India)
Summary. The present experiments revealed a reduction in seedling height and damage at chromosomal level in P i s u m treated with E M S and M M S . F u r t h e r it was observed t h a t GA 3 post-mutagen treatment could reduce this damage considerably b y reducing the number of aberrations as well as chromosomal translocations. The GA 3 t r e a t m e n t also
increased seedling height as well as pollen fertility.
I t is k n o w n t h a t d u p l i c a t i o n s as well as t r a n s l o c a t i o n s do p r o v i d e an i m p o r t a n t source of g e n e t i c v a r i a b i l i t y which m a y y i e l d p l a n t s of useful t y p e s a n d w h i c h could be used f u r t h e r for b r e e d i n g of d e s i r a b l e plants.
For meiotic studies, the floral buds were fixed in Carnoy's fixative (6 parts ethyl alcohol, 3 parts chloroform and t p a r t acetic acid) and squashed in 1% aceto carmine stain.
A large n u m b e r of r e c i p r o c a l t r a n s l o c a t i o n s h a s been i n d u c e d b y v a r i o u s m u t a g e n s . R e c i p r o c a l t r a n s l o c a t i o n s are b y far t h e m o s t c o m m o n s t r u c t u r a l r e a r r a n g e m e n t f o u n d a n d are e a s i l y t r a n s f e r r e d to l a t e r generations. T h e y are n o t o n l y of t h e o r e t i c a l i n t e r e s t b u t are also of p r a c t i c a l a p p l i c a t i o n . H a g b e r g (1964) a n d K o n z a k et al. (t964) h a v e i n d i c a t e d t h e possibilities of usefully e m p l o y i n g reciprocal t r a n s l o c a t i o n s in b r e e d i n g p r o g r a m m e s . L a m m a n d Miravalle (1959) h a v e d e s c r i b e d a t e s t e r set of t r a n s l o c a t i o n s in _Pisum.
T h e d a t a p r e s e n t e d in T a b l e 1 show t h a t t h e r e was a r e d u c t i o n in seedling h e i g h t in E M S - a n d M M S t r e a t e d plants. H o w e v e r , this d a m a g e w a s n e u t r a l i sed w h e n t h e seeds were given p o s t - m u t a g e n t r e a t m e n t of 1000 p p m G A 3. T h e effect of G A 3 was m u c h m o r e e v i d e n t in E M S - t r e a t e d m a t e r i a l t h a n in the M M S . I n E M S - t r e a t e d m a t e r i a l t h e increase in h e i g h t was q u i t e significant a n d even s u r p a s s e d t h e controldby a b o u t 25 p e r cent. T h e increase in h e i g h t in M M S t r e a t m e n t was m o r e or less e q u a l to t h a t of t h e control. G a u r a n d N o t a n i (t960) using maize, H a b e r a n d L u i p p o l d (1960) w i t h w h e a t a n d M a t h u r (196t) w i t h p o t a t o h a v e r e p o r t e d t h e m o d i f i c a t i o n of p h y s i o logical d a m a g e b y m u t a g e n s t h r o u g h p o s t - t r e a t m e n t w i t h gibberellic acid. K u m a r (1967) has r e p o r t e d t h a t G A 3 p o s t - m u t a g e n t r e a t m e n t could r e d u c e t h e c y t o l o g i c a l d a m a g e in g a m m a - i r r a d i a t e d barley 9
T h e p r e s e n t e x p e r i m e n t s were u n d e r t a k e n to induce d u p l i c a t i o n s a n d t r a n s l o c a t i o n s in P i s u m using E M S a n d M M S as well as to see if gibberellic acid (GAs) t r e a t m e n t h a d a n y effect in m o d i f y i n g prod u c t i o n of these d u p l i c a t i o n s a n d t r a n s l o c a t i o n s .
Results and D i s c u s s i o n
Material and Methods Pea seeds of the variety 'Bounville' were presoaked in water for two hours and then treated with one dose each of E M S (0.25%) and M M S (0.0250) for ten hours. The solutions of mutagens were prepared in phosphate buffer adjusted to p H 7.0. At the end of the t0-hour period, the seeds were washed in distilled water for 10 minutes and resoaked in a 1000 p p m solution of gibberellic acid (GAa) for 8 hours. At the end of the t r e a t m e n t the seeds were again washed with distilled water. Four replications, each of 25 treated seeds were sown in wooden flats for recording seedling height. Another set of 100 seeds was sown in the field in 4 plots. The seedling height was recorded after 10 days. For mitotic studies, 25 treated seeds were placed in a petri dish lined with wet filter paper and the root tips were collected and fixed from the germinating seeds. The slides for mitotic studies were prepared b y hydrolysing the root tips with a mixture of 9 parts of 2% aceto orcein and I p a r t of N H C L and then staining with 1 ~o aceto orcein.
Table 1. Seedling injury and milotic studies Mutagenic treatment Control
Mitotic studies x *Seediing height (cms.) bridgesrods dots
% aberrations per cell
5.16 3.40
Nil 49
Nil 14
Nil 27
0.00 0.45
. E M S 0 . 2 5 % + 6.80
35
17
7
0.30
48
25
33
0.53
46
4
32
o.41
E M S 0.25%
GA t o00 p p m M M S 0.025% 3.00 MMS
o.o25~/ +
5.0o
GA
* Seedling height as recorded after 10 days x No. of cells scored -- 200
E f f e c t of Gibberellic Acid on C h r o m o s o l n a l A b e r r a t i o n s in Pisum sativum
Vol. 41, No. 1
19
T a b l e 2. Meiotic studies and pollen [ertility
No. of cells analysed
Treatment
E M S 0.25 % 260 E M S 0.25% + 232 GA t 000 p p m M M S 0.25% 278 M M S 0.025% + GA t96
Metaphase anomalies
Anaphase and teloTotal phase anomalies like -aberrant like rings, chains, dots bridges, laggards, dots, cells fragments, etc. rods, fragments, etc.
% aberration % age fertile per cell pollen grain
46
25
71
0.27
19
27
46
0.20
73
42 22
41 17
83 39
0.30 0.20
62 74
67
T a b l e 3- Metaphase I and I I meiotic anomalies Rings Treatment
Chains
No. of Rings I~ings cells of 3 of 4 analysed chro- chromoso- mosomes rues
E M S 0.25% 260 E M S 0 . 2 5 % + 232 GA 1000 p p m M M S 0.025% 278 MMS0.025% +196 GA 1000 p p m
Rings of 6 Total No. chro- offings mosomes
Chain of 2 chromosomes II
17
Chain of 3 chromosorues
Chain of 4 Total no. chromoso- of chains, rues
FragTotal No. of o 9 ,ments, cells showing 70 aDerranon dots, anomalies per ceu rods, etc.
t5
14
46
0.18
3
14
19
0.08
5
9
3
l
3
l
1
--
2
1
2
--
l0
7
--
17
7
3
1
11
14
42
0.15
1
1
1
3
2
--
1
3
16
22
0.11
T a b l e 4. Anaphase I and I I meiotic anomalies
Treatment
No. analysed of cells
Bridges
Laggards, dots, etc. rods, Total
%age aberrations per cell
E M S 0.25%
260
23
2
25
0.09
E M S 0.25% + GA t000 p p m M M S 0.025% MMS0.025% +GA
232
19
8
27
0.12
278 196
35 14
6 3
41 17
0,15 O.O9
The mitotic studies (Table 1) show that GA a can effectively modify the cytological damage caused by chemical mutagens in the same way as it does with physical mutagens (Kumar, 1967). However, the response to GA a treatment was variable. A marked reduction in the number of bridges and dots was observed in E M S + GAa-treated material. The number of rods was reduced after treatment with M M S + GA 3 but not after treatment with E M S + GA 3. In the meiotic studies (Tables 2, 3 and 4), GAa had more effect on the metaphase than on the anaphase in E M S - t r e a t e d material. The rings and chains were reduced whereas there was no effect on dots and rods. These results corroborate the evidence obtained in the mitotic studies. In M M S - t r e a t e d material, GA 3 treatment had a marked effect both at the metaphase and anaphase stages, the aberrations per cell decreasing considerably at both stages.
From these results it is evident that GA 3 is effective in reducing chromosomal damage, but at the moment it is difficult to indicate how this comes about. Varner (1964) has shown that gibberellic acid controls the synthesis of 0~ amylase in barley endosperm, and that it induces the synthesis of RNA which in turn helps to repair the cytological damage caused by physical and chemical mutagens. Gibberellic acid probably has something to do with the changed structural arrangement of the DNA molecules (after treatment with E M S and M M S ) . Alternatively, increased protein synthesis stimulated by GA a may be helping in the repair mechanism as suggested by Varner
(1964). From the results presented here, it appears that post-mutagen treatment with gibberellic acid could be used with advantage in reducing chromosomal aberrations. Gibberellic acid should be used only if one is interested in reducing the chromosomal aber-
20
V.G. Narsinghani and Sudhir K u m a r : Effect of Gibberellic Acid on ChromosomalAberrations in Pisurn salivurn
r a t i o n s with a view to o b t a i n i n g a n increased n u m b e r of m u t a t i o n s ; it should n o t be used if one is i n t e r e s t e d in obtaining duplications and translocations. T h e d u p l i c a t i o n s a n d t r a n s l o c a t i o n s o b t a i n e d are u n d e r g o i n g f u r t h e r o b s e r v a t i o n s to d e t e r m i n e which chromosomes are i n v o l v e d .
Acknowledgements The authors are grateful to Dr. H. N. Mehrotra, Prolessor and Head of the Department and to Dr. B. K. Srivastava, Director, for the interest taken and facilities provided. The senior author is grateful to the Department of Atomic Energy, Government of India, for the award of a Fellowship as well as the funds for carrying out this project.
Received November 25, 1969 Communicated by H. Stubbe
References 1. Gaur, B . K . , Notani, N . K . : Effect of Gibberellic Acid on barley. Rad. Res. 3, 339--350 (1960). -- 2. Haber, A . H . , Luippold, H . J . : Effects of gibberellin on gamma irradiated wheat. Amer. J. Bot. 47, 140--144 (t960). -- 3. Hagberg, A. : Use of induced translocations in directed production of duplications. Suppl. Rad. Bot. 5, 7 4 1 - 752 (t964). -- 4. Kumar, Sudhir: Effect of Gibberellic Acid o n gamma irradiated barley seeds. Ind. J. Gen. 27, t 54-- 156 (t 967). -- 5. Lamm, R., Miravalle, R. J. : A translocation tester set in Pisum. Hereditas 45, 417 to 440 (1959). -- 6. Mathur, P. B.: Reversal of gamma ray induced dormancy of potato tubers of Gibberellic Acid. Nature, Lond., 19o, 547--548 (1961). -- Nilan, R.A., Konzak, C.F., Wanger, J., Legault, R . R . : The use of induced mutations in Plant Breeding. Supplement to Rad. Bot. 5, 71--89 (1964). -- 8. Varner, J . E . : Gibberellic Acid controlled synthesis of L-amylase in barley endosperm. Plant Physiol. 39, 413-- 415 (1964).
Dr. Sudhir K u m a r Geneticist Department of Genetics and P l a n t Breeding Agricultural Experiment Station University of Udaipur Udaipur, Rajasthan State (India)
