Plant Cell Reports
Plant Cell Reports (1996) 15:437-440
9 Springer-Verlag1996
Somatic embryogenesis in Encephalartos cycadifolius Anna K. Jiiger and Johannes van Staden NU Research Unit for Plant Growth and Development, Department of Botany, University of Natal Pietermaritzburg, P/Bag X01, Scottsville 3209, South Africa Received 10 May 1995/Revised version received 20 July 1995 - Communicated by E Constabel
Abstract. Callus cultures of Encephalartos cycadifolius were established from zygotic embryo explants on a modified B5 medium containing 1 mg 11 2,4-D and 1 mg 11 kinetin. Callus was transferred to media containing various combinations of 2,4-D and kinetin for improvement of somatic embryogenesis. Somatic embryos were produced on media with several growth regulator combinations. The somatic embryos developed from proembryos, which developed long suspensors. A dicotyledonary embryo formed at the distal end of the suspensor. The embryos turned green in light. When transferred to a medium containing 1 mg 1"1ABA the somatic embryos matured. The suspensors desiccated and these embryos rooted when transferred to a medium without phytohormones.
(1992b) working on central American cycads have obtained somatic embryogenesis and organogenesis in Zamia fischeri, Z. furfuracea and Z. pumila. Somatic embryogenesis and morphogenesis have also been reported in Ceratozamia hildae and C. mexicana (Chavez et al. 1992a; Chavez et al. 1992c; Litz et al. 1995). Osborne and van Staden (1987) obtained shoot organogenesis in Stangeria eriopus. This report is the first on in vitro culture and plantlet regeneration via somatic embryogenesis of E.
cycadifolius.
Material and Methods
Explant material Key words: Cyeads Encephalartos cycadifolius Gymnosperm - In vitro culture - Somatic embryogenesis
Abbreviations.ABA:abseisieacid.2,4-D:2,4-diehlorophenoxyacetieacid Introduction Members of the Cycadales, commonly referred to as cycads, have grown on earth for at least 250 million years. They are, therefore, often described as living fossils. Cycads are severely threatened by man's activities which destroy their natural habitats. More threatening is the fact that they have become collectors items.
Encephalartos cycadifollus (Jacq.) Lehm is a small cycad endemic to South Africa. It is found in the mountainous regions of the Eastern Cape Province. Growing at altitudes up to 1800 m it withstands heavy frost during winter. In vitro propagation could present a way of conservation of many of the endangered cycad species. Chavez et al. Correspondence to: A. K. J/iger
Twenty seeds of E. cycadifolius (Jacq.) Lehm from a specimen growing near Bedford in the Eastern Cape were used for excision of explants 12 months after coning. Heavy seeds that sank in water were washed in tap water and then surface-sterilized in 70 % ethanol for 4 min, rinsed and immersed in 1 % sodium hypochlorite for 30 rain and, finally, rinsed three times in sterile water. The sclerotesta was removed with forceps and scalpel. Embryo material was dissected by cutting the megagametophyte longitudinally, whereby the embryo was bisected, also.
Callus initiation Thirty six embryo explants were placed on induction medium, 10 ml in 22 mm x 100 mm tubes, as outlined by Chavez et al. (1992a) supplemented with 1 mg 1"1 2,4-D and 1 mg 1-1 kinetin. The medium contained Gamborg B5 major salts (Gamborg et al. 1968), Murashige and Skoog (1962) minor salts plus vitamins, supplemented with 400 mg 11 glutamine, 100 mg 11 casein hydrolysate, 100 mg 11 arginine, 100 mg 11 asparagine and 60 g 11 sucrose, solidified with 0.8 % agar-agar (Associated Chemical
438 Enterprises c.c., South Africa). The pH prior to autoclaving was 5.7. Cultures were kept in the dark at 26 ~ The cultures were subcultured monthly onto new medium. Cultures were maintained on 40 ml medium in 250 ml jars. To determine the optimal sucrose concentration for callus growth the callus induction medium was supplemented with 2,3,4,5 or 6 % sucrose respectively.
Conditions for embryogenesis To investigate the influence of growth regulators on somatic embryogenesis the medium was supplemented with various combinations of 2,4-D (13, 0.5, 1 and 2 mg 1"1) and kinetin (0, 1, 2 and 3 mg 11). Cultures containing suspensor stage embryos (Fig. 1C), comparable to stage 2 embryos in Picea abies (yon Arnold and Hakman 1988), were transferred to 16h light/8h darkness (13.6 tool m -2s-l). For embryo maturation the medium was supplemented with 1 mg 1I ABA. For roofing a medium without growth regulators was used.
Results
Establishment of callus cultures After 2 weeks in culture the zygotic embryo explants had swollen and callus appeared. The callus was yellow in colour and grew rapidly. Callus was maintained on the induction medium. Best callus growth was recorded with 4 % sucrose. All cultures were subsequently routinely transferred to media with 4 % sucrose.
Somatic embryogenesis After 5 months in culture organized tissue first appeared in the maintenance cultures. To investigate the influence of growth regulators on somatic embryogenesis callus pieces of approximately 1 cm 3 were transferred to media with different combinations of 2,4-D and kinetin. Somatic embryos developed on media at all concentrations of 2,4-D without kinetin and on media with low concentrations of kinetin without 2,4-D or combined with 1 mg 11 2,4-D (Table 1). The highest degree of embryogenesis occurred on media with 1 mg 11 2,4-D and 1 mg 1-1 kinetin after 3 months on the grid. However, after 6 months on the grid, media without growth regulators or with 0.5 mg 1-1 2,4-D yielded the hightest number of embryos (> 100 per jar). After 8 months in culture embryogenic cultures changed appearance and became translucent. Somatic proembryos emerged in the callus (Fig. 1A). The proembryos developed long suspensors, first with a rough surface (Fig. 1B), comparable to stage 1 embryos in P/cea ab/es (yon Arnold and Hakman 1988), later the surface of the suspensors became smooth (Fig. 1C) (Stage 2). A dicotyledonary
Table 1. Influence of hormone composition in media on somatic embryogenesis in E. cycadifolius. Kinetin (rag I"1)
0 0.5 2,4-D (nag 1-1) 1 2
0
1
2E
5E
2
3
4E 3E 1E
10E 1E
Number of embryos per tube 3 month after transfer to grid. E = somatic embryos, - = no response. somatic embryo developed at the distal end of the suspensor (Fig. 1D) (Stage 3). The somatic embryos turned green when exposed to light. For maturation the embryos on suspensors, often still attached to callus, were transferred to a medium containing 1 mg 11 ABA. The suspensor desiccated on the ABA medium (Fig. 1E), and after 5-10 weeks the embryos could be transferred to growth regulator free medium. After 4-5 weeks on the growth-regulator free medium the embryos started to root (Fig. IF). Rooting occurred on 10 % of the embryos. Discussion
Callus of E. cycadifolius grew rapidly on the induction medium containing 1 mg 1"1 2,4-D and 1 mg 1"t kinetin. After 5 months of culture sufficient catlus was obtained to perform experiments with a growth regulator grid. Somatic embryogenesis was induced on media with various concentrations of 2,4-D alone and on media with low concentrations of kinetin alone or combined with 1 mg 1-1 2,4-D. The development of somatic embryos of E. cycadifolius followed the same sequence of somatic embryo development as in Ceratozconia species (Chavez et al. 1992a) and Zamia species (Chavez et al. 1992b). The embryos were transferred to a medium containing ABA for maturation, whereafter they were transferred to growth regulator free medium. In this way we were able to root the embryos. Previously, somatic embryos of C. mexicana and C. hildae (Chavez et al. 1992a; Chavez et al. 1992c; Litz et al. I995) and of Z. pumila (Chavez et al. 1992b) have germinated on a growth regulator free medium. It is, however, still necessary to optimize the maturation and rooting procedures.
439
Fig. 1. Development of somatic embryos orE. cycadlfolius. (A) Callus culture with proembryos. (B) Suspensor with rough surface. (C) Suspensor with smooth surface, (D) Dicotyledonary somatic embryo developing in distal end of snspensor. (E) Somatic embryo with desiccating suspensor, (F) Mature embryo with root.
440 Somatic embryogenesis of cycads holds great promise for mass propagation and conservation of these valuable endangered plants. Acknowledgements. The Foundationfor ResearchDevelopment,Pretoria, is thanked for f'mancial support. Seeds of E. cycadifollus was kindly
donatedby KirstenboschBotanicalGardens,CapeTown.
References Chavez VM, Litz RE, Norstog K (1992a) Plant Cell Tiss. Org. Cult. 30:93-98 Chavez VM, Litz RE, Norstog K (1992b) Plant Cell Tiss. Org. Cult. 30:99-105 Chavez VM, Litz RE, Moon PA, Norstog K (1992c) In Vitro Cell. Dev. BioL 28P:59-63 Gamborg O, Miller RA, Ojima K (1968) Exp. Cell Res. 50, 151-158 Litz RE, Moon PA, Chavez VM (1995) Plant Cell Tiss. Org. Cult. 40:25-31 Murashige T, Skoog F (1962) Physiol. Plant. 15:473-497 Osborne R, van Staden J (1987) HortScience 22:1326 von Arnold S, Hakman I (1988) J. Plant Physiol. 132:164-169
Plant Cell Reports (1996) 15:437-440
9 Springer-Verlag1996
Somatic embryogenesis in Encephalartos cycadifolius Anna K. Jiiger and Johannes van Staden NU Research Unit for Plant Growth and Development, Department of Botany, University of Natal Pietermaritzburg, P/Bag X01, Scottsville 3209, South Africa Received 10 May 1995/Revised version received 20 July 1995 - Communicated by E Constabel
Abstract. Callus cultures of Encephalartos cycadifolius were established from zygotic embryo explants on a modified B5 medium containing 1 mg 11 2,4-D and 1 mg 11 kinetin. Callus was transferred to media containing various combinations of 2,4-D and kinetin for improvement of somatic embryogenesis. Somatic embryos were produced on media with several growth regulator combinations. The somatic embryos developed from proembryos, which developed long suspensors. A dicotyledonary embryo formed at the distal end of the suspensor. The embryos turned green in light. When transferred to a medium containing 1 mg 1"1ABA the somatic embryos matured. The suspensors desiccated and these embryos rooted when transferred to a medium without phytohormones.
(1992b) working on central American cycads have obtained somatic embryogenesis and organogenesis in Zamia fischeri, Z. furfuracea and Z. pumila. Somatic embryogenesis and morphogenesis have also been reported in Ceratozamia hildae and C. mexicana (Chavez et al. 1992a; Chavez et al. 1992c; Litz et al. 1995). Osborne and van Staden (1987) obtained shoot organogenesis in Stangeria eriopus. This report is the first on in vitro culture and plantlet regeneration via somatic embryogenesis of E.
cycadifolius.
Material and Methods
Explant material Key words: Cyeads Encephalartos cycadifolius Gymnosperm - In vitro culture - Somatic embryogenesis
Abbreviations.ABA:abseisieacid.2,4-D:2,4-diehlorophenoxyacetieacid Introduction Members of the Cycadales, commonly referred to as cycads, have grown on earth for at least 250 million years. They are, therefore, often described as living fossils. Cycads are severely threatened by man's activities which destroy their natural habitats. More threatening is the fact that they have become collectors items.
Encephalartos cycadifollus (Jacq.) Lehm is a small cycad endemic to South Africa. It is found in the mountainous regions of the Eastern Cape Province. Growing at altitudes up to 1800 m it withstands heavy frost during winter. In vitro propagation could present a way of conservation of many of the endangered cycad species. Chavez et al. Correspondence to: A. K. J/iger
Twenty seeds of E. cycadifolius (Jacq.) Lehm from a specimen growing near Bedford in the Eastern Cape were used for excision of explants 12 months after coning. Heavy seeds that sank in water were washed in tap water and then surface-sterilized in 70 % ethanol for 4 min, rinsed and immersed in 1 % sodium hypochlorite for 30 rain and, finally, rinsed three times in sterile water. The sclerotesta was removed with forceps and scalpel. Embryo material was dissected by cutting the megagametophyte longitudinally, whereby the embryo was bisected, also.
Callus initiation Thirty six embryo explants were placed on induction medium, 10 ml in 22 mm x 100 mm tubes, as outlined by Chavez et al. (1992a) supplemented with 1 mg 1"1 2,4-D and 1 mg 1-1 kinetin. The medium contained Gamborg B5 major salts (Gamborg et al. 1968), Murashige and Skoog (1962) minor salts plus vitamins, supplemented with 400 mg 11 glutamine, 100 mg 11 casein hydrolysate, 100 mg 11 arginine, 100 mg 11 asparagine and 60 g 11 sucrose, solidified with 0.8 % agar-agar (Associated Chemical
438 Enterprises c.c., South Africa). The pH prior to autoclaving was 5.7. Cultures were kept in the dark at 26 ~ The cultures were subcultured monthly onto new medium. Cultures were maintained on 40 ml medium in 250 ml jars. To determine the optimal sucrose concentration for callus growth the callus induction medium was supplemented with 2,3,4,5 or 6 % sucrose respectively.
Conditions for embryogenesis To investigate the influence of growth regulators on somatic embryogenesis the medium was supplemented with various combinations of 2,4-D (13, 0.5, 1 and 2 mg 1"1) and kinetin (0, 1, 2 and 3 mg 11). Cultures containing suspensor stage embryos (Fig. 1C), comparable to stage 2 embryos in Picea abies (yon Arnold and Hakman 1988), were transferred to 16h light/8h darkness (13.6 tool m -2s-l). For embryo maturation the medium was supplemented with 1 mg 1I ABA. For roofing a medium without growth regulators was used.
Results
Establishment of callus cultures After 2 weeks in culture the zygotic embryo explants had swollen and callus appeared. The callus was yellow in colour and grew rapidly. Callus was maintained on the induction medium. Best callus growth was recorded with 4 % sucrose. All cultures were subsequently routinely transferred to media with 4 % sucrose.
Somatic embryogenesis After 5 months in culture organized tissue first appeared in the maintenance cultures. To investigate the influence of growth regulators on somatic embryogenesis callus pieces of approximately 1 cm 3 were transferred to media with different combinations of 2,4-D and kinetin. Somatic embryos developed on media at all concentrations of 2,4-D without kinetin and on media with low concentrations of kinetin without 2,4-D or combined with 1 mg 11 2,4-D (Table 1). The highest degree of embryogenesis occurred on media with 1 mg 11 2,4-D and 1 mg 1-1 kinetin after 3 months on the grid. However, after 6 months on the grid, media without growth regulators or with 0.5 mg 1-1 2,4-D yielded the hightest number of embryos (> 100 per jar). After 8 months in culture embryogenic cultures changed appearance and became translucent. Somatic proembryos emerged in the callus (Fig. 1A). The proembryos developed long suspensors, first with a rough surface (Fig. 1B), comparable to stage 1 embryos in P/cea ab/es (yon Arnold and Hakman 1988), later the surface of the suspensors became smooth (Fig. 1C) (Stage 2). A dicotyledonary
Table 1. Influence of hormone composition in media on somatic embryogenesis in E. cycadifolius. Kinetin (rag I"1)
0 0.5 2,4-D (nag 1-1) 1 2
0
1
2E
5E
2
3
4E 3E 1E
10E 1E
Number of embryos per tube 3 month after transfer to grid. E = somatic embryos, - = no response. somatic embryo developed at the distal end of the suspensor (Fig. 1D) (Stage 3). The somatic embryos turned green when exposed to light. For maturation the embryos on suspensors, often still attached to callus, were transferred to a medium containing 1 mg 11 ABA. The suspensor desiccated on the ABA medium (Fig. 1E), and after 5-10 weeks the embryos could be transferred to growth regulator free medium. After 4-5 weeks on the growth-regulator free medium the embryos started to root (Fig. IF). Rooting occurred on 10 % of the embryos. Discussion
Callus of E. cycadifolius grew rapidly on the induction medium containing 1 mg 1"1 2,4-D and 1 mg 1"t kinetin. After 5 months of culture sufficient catlus was obtained to perform experiments with a growth regulator grid. Somatic embryogenesis was induced on media with various concentrations of 2,4-D alone and on media with low concentrations of kinetin alone or combined with 1 mg 1-1 2,4-D. The development of somatic embryos of E. cycadifolius followed the same sequence of somatic embryo development as in Ceratozconia species (Chavez et al. 1992a) and Zamia species (Chavez et al. 1992b). The embryos were transferred to a medium containing ABA for maturation, whereafter they were transferred to growth regulator free medium. In this way we were able to root the embryos. Previously, somatic embryos of C. mexicana and C. hildae (Chavez et al. 1992a; Chavez et al. 1992c; Litz et al. I995) and of Z. pumila (Chavez et al. 1992b) have germinated on a growth regulator free medium. It is, however, still necessary to optimize the maturation and rooting procedures.
439
Fig. 1. Development of somatic embryos orE. cycadlfolius. (A) Callus culture with proembryos. (B) Suspensor with rough surface. (C) Suspensor with smooth surface, (D) Dicotyledonary somatic embryo developing in distal end of snspensor. (E) Somatic embryo with desiccating suspensor, (F) Mature embryo with root.
440 Somatic embryogenesis of cycads holds great promise for mass propagation and conservation of these valuable endangered plants. Acknowledgements. The Foundationfor ResearchDevelopment,Pretoria, is thanked for f'mancial support. Seeds of E. cycadifollus was kindly
donatedby KirstenboschBotanicalGardens,CapeTown.
References Chavez VM, Litz RE, Norstog K (1992a) Plant Cell Tiss. Org. Cult. 30:93-98 Chavez VM, Litz RE, Norstog K (1992b) Plant Cell Tiss. Org. Cult. 30:99-105 Chavez VM, Litz RE, Moon PA, Norstog K (1992c) In Vitro Cell. Dev. BioL 28P:59-63 Gamborg O, Miller RA, Ojima K (1968) Exp. Cell Res. 50, 151-158 Litz RE, Moon PA, Chavez VM (1995) Plant Cell Tiss. Org. Cult. 40:25-31 Murashige T, Skoog F (1962) Physiol. Plant. 15:473-497 Osborne R, van Staden J (1987) HortScience 22:1326 von Arnold S, Hakman I (1988) J. Plant Physiol. 132:164-169
