Plant Cell Reports
Plant Cell Reports (1991) 10:325-328
9 Springer-Verlag1991
Effects of L-proline and post-plating temperature treatment on Maize (Zea mays L.) anther culture B. Biiter, J.E. Schmid, and P. Stamp Institute of Plant Sciences, Swiss Federal Institute of Technology (ETH), Eschikon 33, 8315 Lindau, Switzerland Received March 8, 1991/Revised version received June 20, 1991 - Communicated by A.R. Gould
Summary. Comparison of different post-plating temperature regimes with a control treatment (27~ C) revealed that a short-term cold (8/14~ days or 14~ days) as well as a heat treatment (30~ days) increased the production of embryro-likestructures (ELS) from cultured maize anthers. The beneficial effects of short-term cold treatments were magnified 2-3 times when L-proline (PROL) was added to the induction medium (125 - 500 mg/L). In the best treatment (14~ days, 125 mg/L L-proline) one genotype produced 143.5 ELS/100 anthers. Anthers subjected to high temperature (30~ days, 30~ days, 30~ days) generally showed a lower response than did cold treated anthers, although genotypic differences were observed. Regeneration frequency did not appear to be affected by the presence of L-proline in the induction medium. Abbrev.: ELS = Embryo-like-structures PROL = L-proline Key words: Anther culture - Zea mays L. Temperature Treatment - Proline Introduction: Beneficial effects of temperature shock treatments on anther culture productivity were fast described by NITSCH and NORREEL (1973) and NITSCH (1974). Although the mode of action is unknown, temperature shocks are believed to improve androgenesis by diverting normal gametophytic development into a sporophytic pathway leading to the formation of haploid ELS (NITSCH et al., 1982). In maize anther culture, cold treatment of harvested tassels, prior to culture initiation, is commonly used and has been found to increase response (GENOVESI, 1990). Postplating cold treatments (i.e., cold treatments of detached anthers or isolated microspores already in culture) in combination or without tassel pretreatment were also found to increase El,S-production in maize Offprint requests to: B, Bfiter
(NITSCH et al., 1982; DIEU and BECKERT, 1986; DEIMLING et al., 1990; PESCITELLI et al., 1990). Similary, heat treatments (35~ or 30~ of inoculated anthers and microspores have also been used to induce maize androgenesis (COUMANS et al., 1989; GENOVESI, 1990). Recently it was reported, that PROL as a component of the culture medium, can be used to induce a chilling tolerance in maize suspension culture (SONGSTAD et al.~ 1990). Indeed, PROL has been applied as a cyroprotectant for the freeze preservation of cultured maize cells (WHITHERS and KING, 1979). Moreover, PROL seems to be involved with certain aspects of maize embryogenesis. ARMSTRONG and GREEN (1985) demonstrated the essential function of PROL for the establishment and the maintenance of :friable, embryogenic maize callus. To date, very little work has been done relative to the effects of PROL on androgenesis. To our knowledge there is only one report referring to Triticale anther culture suggesting the successful use of PROL in the induction medium (SOZINOV et al., 1981). SCHMID (1988) applied PROL (5 mg/plant) to wheat plants prior to anther culture and observed an increased number of embryogenic anthers. In maize NITSCH et al. (1982) recommended PROL as a component of the induction medium, since it seemed to support root formation of regenerated plantlets. In none of these cases, however, were interactions of PROL with temperature treatments examined. Therefore, the present study was conducted to investigate the effects of post-plating temperature treatments in combination with PROL on maize anther culture. Materials and Methods: The 4 genotypesused in the present study were: Seneca 60 (Robson Seed Farms, NY, USA), H99xFR16 (Dr. Deimling,Universityof Hohenheim, Germany),PA91xFP.16(Dr. Petolino, Dow Elanco, Champaign, USA) and Dan San 91 (This genotype w a s produced in
326 Table 2: Effect of post-plating temperature treatments combined with a L-proline-eontaining (125mg/L= P125) resp. L-proline-free (P 0) induction medium on the production of embryo-like-structures (ELS) and regenerated plants derived from anther culture of 2 maize genotypes (SE=standard error; N.T.--not tested; 200 anthers/genotype and treatment). GENOTYPE:
PA91 X FR16
TEM- ' Induc- No. of ELS PERA- tion Total TURE med. (%-+SE) P0
27~
Fig. 1: Different types of embryo-like-structures (ELS) derived from maize anther culture: a) loose, callus-like; b) root-like; e) compact, yellowish-white, globular; d) soft, pale white; e) ELS with proliferating sections; f) developed plantlet; (a-e: bar=l mm; f: bar=l era). Table 1 (Experiment A): Effect of post-plating temperature treatments on the production of embryo-like-structures (ELS) and regenerated plants derived from anther culture of 2 maize genotypes (SE= standard error). GENO TEMPE- 27~ TYPE RATURE
30~ 4d
30~ 7d
30~ 14d
8/14~ 2d/2d
14oc 4d
Anthers
260
260
500
260
500
8~ 4d
ELS -proSENE- duction/ 4.2 CA 60 100anth. (1.4) (•
4.6 (2.3)
4.6 9.8 5.4 (1.6) (2.7) (1.7)
0
P0
1
P125
35 (17.5+ 7.2) 49 (24.5-+13.0)
0
1
0
2
0
3
Anthers
240
240
240
312
240
312
5.4 (3.2)
4.2 23.8 16.7 (1.5) (9.6) (8.0)
H99 X ELS-proFR 16 duction/ 3.8 100anth. (1.7) (+SE) Regen. plants
0
1
0
2
1
14oc 4d
30~ 4d
1988 by crossing the inbred Pa-Tang-Be and the F1 of the inbred Shao-Pa-Tang and the inbred Shni-Be on the experimental station of ETH in Eschikon, Switzerland. The inbred and the Ft were provided by Dr. Li Dawei, Institute of Genetics, Beijing, China.). All donor plants were grown in the greenhouse during the period JuneNovember 1990 in Eschikon, Switzerland (25~176 + 3~ 16 hrs light). Tassels were harvested prior to their emergence from the whorl. After a cold pretreatment (6-80C, 10-14 days), tassel segments, with anthers containing late-uninucleate or eady-binudeate microspores, were selected, and surface sterilized (0.6 % v/v sodium hytx~hloride for 15 min.). Anthers were plated m 20 x 60 mm petfi dishes with 10 mL aliquots of induction medium. In order to minimize differences between the donor plants, the required number of anthers for each replication was collected from one or two tassels
84 (42.0-+12.1) 139 (69.5+20.3)
P 0
0
3
0
N.T.
P 125 17 (8.5 • 2.6) P 125 61 (30.5_+ 9.0)
2
N.T.
0
P 0 30~ 14
Plant Cell Reports (1991) 10:325-328
9 Springer-Verlag1991
Effects of L-proline and post-plating temperature treatment on Maize (Zea mays L.) anther culture B. Biiter, J.E. Schmid, and P. Stamp Institute of Plant Sciences, Swiss Federal Institute of Technology (ETH), Eschikon 33, 8315 Lindau, Switzerland Received March 8, 1991/Revised version received June 20, 1991 - Communicated by A.R. Gould
Summary. Comparison of different post-plating temperature regimes with a control treatment (27~ C) revealed that a short-term cold (8/14~ days or 14~ days) as well as a heat treatment (30~ days) increased the production of embryro-likestructures (ELS) from cultured maize anthers. The beneficial effects of short-term cold treatments were magnified 2-3 times when L-proline (PROL) was added to the induction medium (125 - 500 mg/L). In the best treatment (14~ days, 125 mg/L L-proline) one genotype produced 143.5 ELS/100 anthers. Anthers subjected to high temperature (30~ days, 30~ days, 30~ days) generally showed a lower response than did cold treated anthers, although genotypic differences were observed. Regeneration frequency did not appear to be affected by the presence of L-proline in the induction medium. Abbrev.: ELS = Embryo-like-structures PROL = L-proline Key words: Anther culture - Zea mays L. Temperature Treatment - Proline Introduction: Beneficial effects of temperature shock treatments on anther culture productivity were fast described by NITSCH and NORREEL (1973) and NITSCH (1974). Although the mode of action is unknown, temperature shocks are believed to improve androgenesis by diverting normal gametophytic development into a sporophytic pathway leading to the formation of haploid ELS (NITSCH et al., 1982). In maize anther culture, cold treatment of harvested tassels, prior to culture initiation, is commonly used and has been found to increase response (GENOVESI, 1990). Postplating cold treatments (i.e., cold treatments of detached anthers or isolated microspores already in culture) in combination or without tassel pretreatment were also found to increase El,S-production in maize Offprint requests to: B, Bfiter
(NITSCH et al., 1982; DIEU and BECKERT, 1986; DEIMLING et al., 1990; PESCITELLI et al., 1990). Similary, heat treatments (35~ or 30~ of inoculated anthers and microspores have also been used to induce maize androgenesis (COUMANS et al., 1989; GENOVESI, 1990). Recently it was reported, that PROL as a component of the culture medium, can be used to induce a chilling tolerance in maize suspension culture (SONGSTAD et al.~ 1990). Indeed, PROL has been applied as a cyroprotectant for the freeze preservation of cultured maize cells (WHITHERS and KING, 1979). Moreover, PROL seems to be involved with certain aspects of maize embryogenesis. ARMSTRONG and GREEN (1985) demonstrated the essential function of PROL for the establishment and the maintenance of :friable, embryogenic maize callus. To date, very little work has been done relative to the effects of PROL on androgenesis. To our knowledge there is only one report referring to Triticale anther culture suggesting the successful use of PROL in the induction medium (SOZINOV et al., 1981). SCHMID (1988) applied PROL (5 mg/plant) to wheat plants prior to anther culture and observed an increased number of embryogenic anthers. In maize NITSCH et al. (1982) recommended PROL as a component of the induction medium, since it seemed to support root formation of regenerated plantlets. In none of these cases, however, were interactions of PROL with temperature treatments examined. Therefore, the present study was conducted to investigate the effects of post-plating temperature treatments in combination with PROL on maize anther culture. Materials and Methods: The 4 genotypesused in the present study were: Seneca 60 (Robson Seed Farms, NY, USA), H99xFR16 (Dr. Deimling,Universityof Hohenheim, Germany),PA91xFP.16(Dr. Petolino, Dow Elanco, Champaign, USA) and Dan San 91 (This genotype w a s produced in
326 Table 2: Effect of post-plating temperature treatments combined with a L-proline-eontaining (125mg/L= P125) resp. L-proline-free (P 0) induction medium on the production of embryo-like-structures (ELS) and regenerated plants derived from anther culture of 2 maize genotypes (SE=standard error; N.T.--not tested; 200 anthers/genotype and treatment). GENOTYPE:
PA91 X FR16
TEM- ' Induc- No. of ELS PERA- tion Total TURE med. (%-+SE) P0
27~
Fig. 1: Different types of embryo-like-structures (ELS) derived from maize anther culture: a) loose, callus-like; b) root-like; e) compact, yellowish-white, globular; d) soft, pale white; e) ELS with proliferating sections; f) developed plantlet; (a-e: bar=l mm; f: bar=l era). Table 1 (Experiment A): Effect of post-plating temperature treatments on the production of embryo-like-structures (ELS) and regenerated plants derived from anther culture of 2 maize genotypes (SE= standard error). GENO TEMPE- 27~ TYPE RATURE
30~ 4d
30~ 7d
30~ 14d
8/14~ 2d/2d
14oc 4d
Anthers
260
260
500
260
500
8~ 4d
ELS -proSENE- duction/ 4.2 CA 60 100anth. (1.4) (•
4.6 (2.3)
4.6 9.8 5.4 (1.6) (2.7) (1.7)
0
P0
1
P125
35 (17.5+ 7.2) 49 (24.5-+13.0)
0
1
0
2
0
3
Anthers
240
240
240
312
240
312
5.4 (3.2)
4.2 23.8 16.7 (1.5) (9.6) (8.0)
H99 X ELS-proFR 16 duction/ 3.8 100anth. (1.7) (+SE) Regen. plants
0
1
0
2
1
14oc 4d
30~ 4d
1988 by crossing the inbred Pa-Tang-Be and the F1 of the inbred Shao-Pa-Tang and the inbred Shni-Be on the experimental station of ETH in Eschikon, Switzerland. The inbred and the Ft were provided by Dr. Li Dawei, Institute of Genetics, Beijing, China.). All donor plants were grown in the greenhouse during the period JuneNovember 1990 in Eschikon, Switzerland (25~176 + 3~ 16 hrs light). Tassels were harvested prior to their emergence from the whorl. After a cold pretreatment (6-80C, 10-14 days), tassel segments, with anthers containing late-uninucleate or eady-binudeate microspores, were selected, and surface sterilized (0.6 % v/v sodium hytx~hloride for 15 min.). Anthers were plated m 20 x 60 mm petfi dishes with 10 mL aliquots of induction medium. In order to minimize differences between the donor plants, the required number of anthers for each replication was collected from one or two tassels
84 (42.0-+12.1) 139 (69.5+20.3)
P 0
0
3
0
N.T.
P 125 17 (8.5 • 2.6) P 125 61 (30.5_+ 9.0)
2
N.T.
0
P 0 30~ 14
