Plant Cell Reports
Plant Cell Reports (1983) 2:198-200
© Springer-Verlag 1983
Haploid Plants from in vitro Anther Culture of A nnona s q u a m o s a Linn * S. Nair, P. K. Gupta, and A. F. Mascarenhas ** Biochemistry Division, National Chemical Laboratory, Pune 411 008, India Received April 20, 1983 / May 27, 1983 - Communicated by F. Constabel
ABSTRACT
MATERIALS AND METHODS
Haploid plants were induced from anther callus of Annona squamosa Linn. (Custard apple) on a Nitsch basal medium supplemented with 6-benzylaminopurine and naphthalene acetic acid. When naphthalene acetic acid was replaced by indole-3acetic acid only m u l t i p l e shoots were obtained. Pretreatment ( c h i l l i n g , c e n t r i f u g a t i o n , reduced atmospheric pressure e t c . ) of the flowers was not e f f e c t i v e but dissection of the flowers in a suspension of activated charcoal and sucrose was found essential. The anthers required an i n i t i a l dark period and a high sucrose medium followed by l i g h t and lowered sucrose l e v e l s . Root t i p squashes of the regenerated p l a n t l e t s revealed the haploid (n = 7) nature of the p l a n t l e t s . ABBREVIATIONS BAP 2,4-D IAA NAA
S t e r i l i z a t i o n of plant material Anthers from flower buds (length 1.0 - 1.5 cm) of Annona squamosa containing pollen grains at miduninucleate to late uninucleate stage were used as the source of inoculum. The flower buds were surface s t e r i l i z e d with 0.02% HgCI2 f o r I0 min and then washed free of the s t e r i l a n t . Media composition The basal medium (N) used in a l l experiments was that of Nitsch and Nitsch (Nitsch and Nitsch, 1969). The hormone and other supplements added to the basal medium were as follows : N-I
6-benzylaminopurine 2,4-dichlorophenoxyacetic acid indole-3-acetic acid oC-naphthalene acetic acid
INTRODUCTION Androgenesis has been reported in many herbaceous species belonging to d i f f e r e n t genera and f a m i l i e s of angiosperms (Maheshwari et a l . , 1982). The a v a i l a b i l i t y of haploids has special significance f o r the genetic improvement of f r u i t trees where breeding is made d i f f i c u l t by long generation i n t e r v a l s , by the h i g h l y heterozygous nature of most f r u i t plants and by factors such as parthenocarpy and s e l f - i n c o m p a t i b i l i t y (Rajasekaran and M u l l i n s , 1979). Reports on anther cultures from f r u i t trees are s t i l l very few. Either callus or a few pollen d i v i s i o n s were obtained from anther cultures of d i f f e r e n t species of Prunus and Malus (Maheshwari et a l . , 1982). With V i t i s and Prunus cerasus p l a n t l e t s developed which were d i p l o i d ~-jasekaran and M u l l i n s , 1979; S e i r l i s et a l . , 1979). With Coffea arabica haploid callus and subsequent formation of embryoids were observed (Sharp et a l . 1973).
* Communication No. 3187 ** To whom all correspondence may be addressed
N-2 N-3 N-4 N-5
N supplemented with L-serine (I00 mg/l), 2,4-D (3 mg/l), IAA (0.5 mg/l), Sucrose N plus IAA (5 mg/l) N supplemented with (2 mg/l) N plus BAP (2 mg/l) N l i q u i d medium
glutamine (800 mg/l), i n o s i t o l (I000 mg/l), (2 mg/l) k i n e t i n 6%. NAA (I mg/l) and BAP and IAA (0.I mg/l)
N-2, N-3, N-4 and N-5 were supplied with 2% sucrose. In some experiments described in the t e x t N-2 medium with 6% sucrose was also used. A l l semi-solid media were gelled with 0.8% agar. Al! chemicals used were obtained from B r i t i s h Drug House, Sigma, Difco or E. Merck. Inoculation and incubation of anthers Dissection of anthers was carried out in p e t r i dishes (8 cm d i a . ) containing I0 ml of a solution of 6% sucrose and 0.25% activated charcoal and l a t e r inoculated into 20 ml agar medium (N-I) in petri dishes. Th~se were incubated in the dark for 7 days at 25 + 2 C before transfer to l i g h t (18 h l i g h t of lO00 lux and 6 h dark) supplied by cool, daylight fluorescent tubes. After 25 - 30 days, counts of the various responses like swellin~, greening, etc. were made and the results expressed
199 as percentages (20 anther groups were inoculated per p e t r i dish and one group of anthers per tube). In each experiment 5 r e p l i c a t e s were used. The conditions f o r p l a n t l e t formation using fresh anthers were repeated 3 times and found to be reproducible. Cytological studies Chromosome counts were made from root t i p squashes of the regenerated anther p l a n t l e t s using the standard a c e t i c - o r c e i n staining method (Sharma and Sharma, 1980). RESULTS AND DISCUSSION In i n i t i a l experiments single anthers and anther groups were cultured on N-I medium but the former did not respond (Table I , No.I). Further experiments were therefore carried out using anther groups (I0 - 12 anthers per group) as inoculum. D i f f e r e n t pretreatments ( c h i l l i n g , c e n t r i f u g a t i o n , reduced atmospheric pressure e t c . ) of flowers or anthers was found i n e f f e c t i v e unlike many e a r l i e r reports (Xu and Sunderland, 1981; Tyagi et a l . , 1979). I f anthers were d i r e c t l y inoculated into N-I medium over 80% blackened and died a f t e r i n o c u l a t i o n . This could be prevented only by dissection of the anthers from the flowers in a solution of sucrose and activated charcoal before f u r t h e r i n o c u l a t i o n i n t o petri dishes. By t h i s method the addition of antiphenolic substances l i k e activated charcoal, p o l y v i n y l p y r r o l i d o n e etc. i n t o the medium reported e a r l i e r f o r anther cultures was unnecessary (Anagnostakis, 1974; Tyagi et a l . , 1981). The e f f e c t is possibly due to adsorption of phenolic substances that emanate from the anthers during dissection which i n h i b i t the development of pollen grains. Table
l Response of anthers to various media
No.
Medium
During the one-week incubation in dark a s l i g h t swelling was observed in some of the anther groups. Transfer of these p e t r i dishes at t h i s stage to l i g h t resulted in f u r t h e r swelling in 50% of the anthers (Fig. I ) which also turned green (Table I , No.2). A s i m i l a r response regarding an i n i t i a l dark period followed by l i g h t was also found necessary for species l i k e Nicotiana and Datura (Sunderland and Roberts, 1977; Sangwan-Norreel, 1977). A f t e r about 3-4 days in l i g h t the i n d i v i d u a l anther groups were transferred to tubes with N-2 medium and containing e i t h e r 2 or 6% sucrose and incubated in l i g h t , since they blackened and died i f l e f t longer on N-l'medium. On N-2 medium bursting of the swollen anthers and production of white to l i g h t grey, granular, f r i a b l e c a l l u s occured a f t e r about two weeks (Fig. 2).
Fig.l Anther gmoups showing swollen anthers on N-I medium (a 14 day old culture).
Response of single anthers/anther groups
Percentage of single anthers/anther groups showing p o s i t i v e response
I.
N-I (6% suc)
Anthers turn brown and die
2.
N-I (6% suc)
Swelling and greening
50
3.
N-2 (6% suc)
Bursting of swollen anthers and production of callus
25
4.
N-2 (2% suc)
Bursting of swollen anthers and production of callus
80
5.
N-3 (2% suc)
Regeneration of p l a n t l e t s
6.
N-4 (2% suc)
I,i u l t i p l e shoot formation
The anthers used in these experiments were dissected in a solution containing 6% sucrose and 0.25% activated charcoal. In No.l anthers were inoculated s i n g l y (20 anthers per petri d i s h ) . In No.2, 20 groups of anthers (I0 - 12 anthers per group) were inoculated per p e t r i dish. In Nos.3-6 one group of anthers was transferred from the p e t r i dish to one tube. The responses are the average values of c u l t u r e vessels. Culture conditions : 25 ~ 2 C, 18 h photoperiod (I000 l u x ) .
Fig.2 Granular and f r i a b l e anther callus on N-2 medium (a 30 day old culture).
0
5 I0 The percentage of anthers that callused was about 80% on N-2 medium containing 2% sucrose, and only only 25% on N-2 medium in which the sucrose level was maintained at 6% as in the i n i t i a l medium (Table I , Nos. 3 and 4). A s i m i l a r observation was made with anthers of Brassica campestris and Solarium tuberosum which were cultured on high sucrose media i n i t i a l l y followed by t r a n s f e r to lower l e v e l s of sucrose for optimal post-induction growth ( K e l l e r et a l . , 1975; Sopory, 1979). The reason f o r a requirement of higher concentration of sucrose is not c l e a r , although an osmoregulatory role has been suggested (Binding, 1972).
200 For regeneration, anther c a l l i were then subcultured into two d i f f e r e n t media: N-3, containing 8AP and NAA and N-4, containing BAP and IAA. On N-3 medium 5% of c a l l i masses d i r e c t l y regenerated p l a n t l e t s , while the callus on N-4 medium produced multiple shoots in 10% of the cultures (Fig. 3). The regenerated p l a n t l e t s were transferred to N-5 medium for further growth. The root t i p squashes of the regenerated p l a n t l e t s on N-3 medium showed half the somatic number of chromosomes (n = 7, Fig. 4 ) . , Studies to increase the percentage of haploid p l a n t l e t s and t h e i r survival in f i e l d are in progress. As f a r as we are aware this is the f i r s t report on the induction of haploid plantlets by anther culture from f r u i t trees.
ACKNOWLEDGEMENTS We thank Prof. D. P. 8hore, Department of Horticulture, Agricultural College, Pune, for permission to c o l l e c t flower buds from the custard apple trees, Dr. C. P. Joshi for cytological studies and Mr. V. A. Padale for photography. REFERENCES I . Anagnostakis SL (1974) Planta 115:281-283. 2. Binding H (1972) Nature 237:283-285. 3. Keller WA, Rajhathy T, Lacapra J (1975) Can J Genet Cytol 17:655-666. 4. Maheshwari SC, Rashid A, Tyagi AK (1982) Amer J Bot 69:865-879. 5. Nitsch JP, Nitsch C (1969) Science 163:85-87. 6. Rajasekaran K, Mullins MG (1979) J Expt Bot 30:399-407. 7. Sangwan-Norreel BS (1977) J Expt Bot 28:843852. 8. S e i r l i s G, Mouras A, Salesses G (1979) Ann Am'elior Plantes 29:145-161. g. Sharma AK, Sharma A (1980) Chromosome techniques: Theory and Practice, Butterworths and Co., London p. 169-170. I0. Sharp WR, Caldas LS, Crocomo OJ, Monaco LC, Carvalho A (1973) Phyton 31:67-74. I I . Sopo~ SK (1979) Can J Bot 57:2691-2694. 12. Sunderland N, Roberts M (1977) Nature 270: 236-238. 13. Tyagi AK, Rashid A, Maheshwari SC (1979) Protoplasma 99:11-17.
Fig.3 Multiple shoots from anther callus on N-4 medium (a 60 day old culture)
Fig.4 A cell from the root t i p showing haploid number (n = 7) of chromosomes (magnification: 1260 times)
14. Tyagi AK, Rashid A, Maheshwari SC (1981) Physiol Plant 53:405-406. 15. Xu ZH, Sunderland N (1981) Plant Sci Lett 23:161-168.
Plant Cell Reports (1983) 2:198-200
© Springer-Verlag 1983
Haploid Plants from in vitro Anther Culture of A nnona s q u a m o s a Linn * S. Nair, P. K. Gupta, and A. F. Mascarenhas ** Biochemistry Division, National Chemical Laboratory, Pune 411 008, India Received April 20, 1983 / May 27, 1983 - Communicated by F. Constabel
ABSTRACT
MATERIALS AND METHODS
Haploid plants were induced from anther callus of Annona squamosa Linn. (Custard apple) on a Nitsch basal medium supplemented with 6-benzylaminopurine and naphthalene acetic acid. When naphthalene acetic acid was replaced by indole-3acetic acid only m u l t i p l e shoots were obtained. Pretreatment ( c h i l l i n g , c e n t r i f u g a t i o n , reduced atmospheric pressure e t c . ) of the flowers was not e f f e c t i v e but dissection of the flowers in a suspension of activated charcoal and sucrose was found essential. The anthers required an i n i t i a l dark period and a high sucrose medium followed by l i g h t and lowered sucrose l e v e l s . Root t i p squashes of the regenerated p l a n t l e t s revealed the haploid (n = 7) nature of the p l a n t l e t s . ABBREVIATIONS BAP 2,4-D IAA NAA
S t e r i l i z a t i o n of plant material Anthers from flower buds (length 1.0 - 1.5 cm) of Annona squamosa containing pollen grains at miduninucleate to late uninucleate stage were used as the source of inoculum. The flower buds were surface s t e r i l i z e d with 0.02% HgCI2 f o r I0 min and then washed free of the s t e r i l a n t . Media composition The basal medium (N) used in a l l experiments was that of Nitsch and Nitsch (Nitsch and Nitsch, 1969). The hormone and other supplements added to the basal medium were as follows : N-I
6-benzylaminopurine 2,4-dichlorophenoxyacetic acid indole-3-acetic acid oC-naphthalene acetic acid
INTRODUCTION Androgenesis has been reported in many herbaceous species belonging to d i f f e r e n t genera and f a m i l i e s of angiosperms (Maheshwari et a l . , 1982). The a v a i l a b i l i t y of haploids has special significance f o r the genetic improvement of f r u i t trees where breeding is made d i f f i c u l t by long generation i n t e r v a l s , by the h i g h l y heterozygous nature of most f r u i t plants and by factors such as parthenocarpy and s e l f - i n c o m p a t i b i l i t y (Rajasekaran and M u l l i n s , 1979). Reports on anther cultures from f r u i t trees are s t i l l very few. Either callus or a few pollen d i v i s i o n s were obtained from anther cultures of d i f f e r e n t species of Prunus and Malus (Maheshwari et a l . , 1982). With V i t i s and Prunus cerasus p l a n t l e t s developed which were d i p l o i d ~-jasekaran and M u l l i n s , 1979; S e i r l i s et a l . , 1979). With Coffea arabica haploid callus and subsequent formation of embryoids were observed (Sharp et a l . 1973).
* Communication No. 3187 ** To whom all correspondence may be addressed
N-2 N-3 N-4 N-5
N supplemented with L-serine (I00 mg/l), 2,4-D (3 mg/l), IAA (0.5 mg/l), Sucrose N plus IAA (5 mg/l) N supplemented with (2 mg/l) N plus BAP (2 mg/l) N l i q u i d medium
glutamine (800 mg/l), i n o s i t o l (I000 mg/l), (2 mg/l) k i n e t i n 6%. NAA (I mg/l) and BAP and IAA (0.I mg/l)
N-2, N-3, N-4 and N-5 were supplied with 2% sucrose. In some experiments described in the t e x t N-2 medium with 6% sucrose was also used. A l l semi-solid media were gelled with 0.8% agar. Al! chemicals used were obtained from B r i t i s h Drug House, Sigma, Difco or E. Merck. Inoculation and incubation of anthers Dissection of anthers was carried out in p e t r i dishes (8 cm d i a . ) containing I0 ml of a solution of 6% sucrose and 0.25% activated charcoal and l a t e r inoculated into 20 ml agar medium (N-I) in petri dishes. Th~se were incubated in the dark for 7 days at 25 + 2 C before transfer to l i g h t (18 h l i g h t of lO00 lux and 6 h dark) supplied by cool, daylight fluorescent tubes. After 25 - 30 days, counts of the various responses like swellin~, greening, etc. were made and the results expressed
199 as percentages (20 anther groups were inoculated per p e t r i dish and one group of anthers per tube). In each experiment 5 r e p l i c a t e s were used. The conditions f o r p l a n t l e t formation using fresh anthers were repeated 3 times and found to be reproducible. Cytological studies Chromosome counts were made from root t i p squashes of the regenerated anther p l a n t l e t s using the standard a c e t i c - o r c e i n staining method (Sharma and Sharma, 1980). RESULTS AND DISCUSSION In i n i t i a l experiments single anthers and anther groups were cultured on N-I medium but the former did not respond (Table I , No.I). Further experiments were therefore carried out using anther groups (I0 - 12 anthers per group) as inoculum. D i f f e r e n t pretreatments ( c h i l l i n g , c e n t r i f u g a t i o n , reduced atmospheric pressure e t c . ) of flowers or anthers was found i n e f f e c t i v e unlike many e a r l i e r reports (Xu and Sunderland, 1981; Tyagi et a l . , 1979). I f anthers were d i r e c t l y inoculated into N-I medium over 80% blackened and died a f t e r i n o c u l a t i o n . This could be prevented only by dissection of the anthers from the flowers in a solution of sucrose and activated charcoal before f u r t h e r i n o c u l a t i o n i n t o petri dishes. By t h i s method the addition of antiphenolic substances l i k e activated charcoal, p o l y v i n y l p y r r o l i d o n e etc. i n t o the medium reported e a r l i e r f o r anther cultures was unnecessary (Anagnostakis, 1974; Tyagi et a l . , 1981). The e f f e c t is possibly due to adsorption of phenolic substances that emanate from the anthers during dissection which i n h i b i t the development of pollen grains. Table
l Response of anthers to various media
No.
Medium
During the one-week incubation in dark a s l i g h t swelling was observed in some of the anther groups. Transfer of these p e t r i dishes at t h i s stage to l i g h t resulted in f u r t h e r swelling in 50% of the anthers (Fig. I ) which also turned green (Table I , No.2). A s i m i l a r response regarding an i n i t i a l dark period followed by l i g h t was also found necessary for species l i k e Nicotiana and Datura (Sunderland and Roberts, 1977; Sangwan-Norreel, 1977). A f t e r about 3-4 days in l i g h t the i n d i v i d u a l anther groups were transferred to tubes with N-2 medium and containing e i t h e r 2 or 6% sucrose and incubated in l i g h t , since they blackened and died i f l e f t longer on N-l'medium. On N-2 medium bursting of the swollen anthers and production of white to l i g h t grey, granular, f r i a b l e c a l l u s occured a f t e r about two weeks (Fig. 2).
Fig.l Anther gmoups showing swollen anthers on N-I medium (a 14 day old culture).
Response of single anthers/anther groups
Percentage of single anthers/anther groups showing p o s i t i v e response
I.
N-I (6% suc)
Anthers turn brown and die
2.
N-I (6% suc)
Swelling and greening
50
3.
N-2 (6% suc)
Bursting of swollen anthers and production of callus
25
4.
N-2 (2% suc)
Bursting of swollen anthers and production of callus
80
5.
N-3 (2% suc)
Regeneration of p l a n t l e t s
6.
N-4 (2% suc)
I,i u l t i p l e shoot formation
The anthers used in these experiments were dissected in a solution containing 6% sucrose and 0.25% activated charcoal. In No.l anthers were inoculated s i n g l y (20 anthers per petri d i s h ) . In No.2, 20 groups of anthers (I0 - 12 anthers per group) were inoculated per p e t r i dish. In Nos.3-6 one group of anthers was transferred from the p e t r i dish to one tube. The responses are the average values of c u l t u r e vessels. Culture conditions : 25 ~ 2 C, 18 h photoperiod (I000 l u x ) .
Fig.2 Granular and f r i a b l e anther callus on N-2 medium (a 30 day old culture).
0
5 I0 The percentage of anthers that callused was about 80% on N-2 medium containing 2% sucrose, and only only 25% on N-2 medium in which the sucrose level was maintained at 6% as in the i n i t i a l medium (Table I , Nos. 3 and 4). A s i m i l a r observation was made with anthers of Brassica campestris and Solarium tuberosum which were cultured on high sucrose media i n i t i a l l y followed by t r a n s f e r to lower l e v e l s of sucrose for optimal post-induction growth ( K e l l e r et a l . , 1975; Sopory, 1979). The reason f o r a requirement of higher concentration of sucrose is not c l e a r , although an osmoregulatory role has been suggested (Binding, 1972).
200 For regeneration, anther c a l l i were then subcultured into two d i f f e r e n t media: N-3, containing 8AP and NAA and N-4, containing BAP and IAA. On N-3 medium 5% of c a l l i masses d i r e c t l y regenerated p l a n t l e t s , while the callus on N-4 medium produced multiple shoots in 10% of the cultures (Fig. 3). The regenerated p l a n t l e t s were transferred to N-5 medium for further growth. The root t i p squashes of the regenerated p l a n t l e t s on N-3 medium showed half the somatic number of chromosomes (n = 7, Fig. 4 ) . , Studies to increase the percentage of haploid p l a n t l e t s and t h e i r survival in f i e l d are in progress. As f a r as we are aware this is the f i r s t report on the induction of haploid plantlets by anther culture from f r u i t trees.
ACKNOWLEDGEMENTS We thank Prof. D. P. 8hore, Department of Horticulture, Agricultural College, Pune, for permission to c o l l e c t flower buds from the custard apple trees, Dr. C. P. Joshi for cytological studies and Mr. V. A. Padale for photography. REFERENCES I . Anagnostakis SL (1974) Planta 115:281-283. 2. Binding H (1972) Nature 237:283-285. 3. Keller WA, Rajhathy T, Lacapra J (1975) Can J Genet Cytol 17:655-666. 4. Maheshwari SC, Rashid A, Tyagi AK (1982) Amer J Bot 69:865-879. 5. Nitsch JP, Nitsch C (1969) Science 163:85-87. 6. Rajasekaran K, Mullins MG (1979) J Expt Bot 30:399-407. 7. Sangwan-Norreel BS (1977) J Expt Bot 28:843852. 8. S e i r l i s G, Mouras A, Salesses G (1979) Ann Am'elior Plantes 29:145-161. g. Sharma AK, Sharma A (1980) Chromosome techniques: Theory and Practice, Butterworths and Co., London p. 169-170. I0. Sharp WR, Caldas LS, Crocomo OJ, Monaco LC, Carvalho A (1973) Phyton 31:67-74. I I . Sopo~ SK (1979) Can J Bot 57:2691-2694. 12. Sunderland N, Roberts M (1977) Nature 270: 236-238. 13. Tyagi AK, Rashid A, Maheshwari SC (1979) Protoplasma 99:11-17.
Fig.3 Multiple shoots from anther callus on N-4 medium (a 60 day old culture)
Fig.4 A cell from the root t i p showing haploid number (n = 7) of chromosomes (magnification: 1260 times)
14. Tyagi AK, Rashid A, Maheshwari SC (1981) Physiol Plant 53:405-406. 15. Xu ZH, Sunderland N (1981) Plant Sci Lett 23:161-168.
