PlantCell Reports
Plant Cell Reports (1996) 16:137-141
© Springer-Verlag1996
Primary callus as source of totipotent barley (Hordeum vulgare L.) protoplasts Annette Sti~ldt *~ Xiao-Hui Wang *' **, and Horst Ltirz Zentrum ftir Angewandte Molekularbiologie der Pflanzen (AMP II), Institut fiir Allgemeine Botanik, Universit/it Hamburg, Ohnhorststr. 18, D-22609 Hamburg, Germany * These authors have contributed equally to the work presented ** Present address: Department of Biology, Massachusetts Institute of Technology, Building 68-617, 77 Massachusetts Avenue, Cambridge, MA 02139, USA Received 15 May 1996/Revised version received 24 May 1996 - Communicated by K. Glimelius
Summary Primary callus of barley (Hordeum vulgare L.) derived from scutella (cv. 'Dissa') and anthers (cv. 'Igri') was used for protoplast isolation and plant regeneration. The protoplasts were embedded in agarose and cultured with nurse cells. The plating efficiency varied from 0,1% to 0.7%. Shoots regenerated from the developing callus. Plantlets were transferred to soil and cultivated in the greenhouse three to five months after protoplast isolation. All plants were normal in morphology, and most of them flowered and set seeds. Key words: Protoplasts - Hordeum vulgare L. - nurse culture plant regeneration - primary callus
Abbreviations: 2,4-D: 2,4-dichlorophenoxyacetic acid, BAP: 6-benylaminopurine
(Jhhne et al 1991 a,b, Funatsuki et al, 1992, Golds et al. 1994, Kihara and Funatsuki 1995). This indicates that plant regeneration from barley protoplasts still remains a problem. As an alternative, it has been suggested recently to use primary callus as a source for protoplast isolation. In the case of barley, primary callus can be produced from immature embryos (Kihara and Funatsuld 1995), microspores (Salmenkallio-Martilla and Kauppinen 1995) or anthers. The obvious advantage of this material lies in the shortened tissue culture period and, subsequently, in a reduced risk of somaclonal variation. We present here two rapid regeneration systems for protoplasts from primary callus derived from either young scutella or anthers of barley.
Material and methods Plant material. The spring type barley cultivar 'Dissa'
Introduction Protoplasts represent a useful system for plant genetic manipulations such as somatic hybridisation, cytoplasmic recombination or direct gene transfer. For this, an efficient plant regeneration system from isolated protoplasts is a prerequisite. The major source for protoplasts of cereal crops are embryogenic cell suspensions (reviews: Vasil 1992, Krautwig and L6rz 1995). The establishment of suspension cultures producing a high yield of viable protoplasts is very laborious and strongly genotypedependent. Additional problems arise during the long period of culture with a decrease in morphogenic capacity (J~me et al. 1991a,b) and the occurrence of somaclonal variation (Karp et al. 1987, Wang et al, 1992). Up to now, there are only few reports on efficient and reliable regeneration of fertile barley plants from suspension culture-derived protoplasts Correspondence to: H. L6rz
was used for the initiation of primary callus from immature embryos. For callus induction from anthers the winter-type 'Igri' was used. Barley plants of the cultivar 'Gimpel' were grown for the isolation of microspores which were added as nurse cells. Induction o f primary callus. Immature embryos were
harvested at a length of 0.5 to 1.0 mm. The embryo axes were removed and the scutella cultured upside down on L2 medium (Lazzeri et al. 1991) supplemented with 1 mg/1 2,4-D. Spiklets containing pollen at the mid-uninucleate stage were pretreated at 4°C in the dark for 14 to 21 days. The anthers were cultured on L3 medium (Lazzeri et al. 1991) supplemented with 1 mg/1 2,4-D and 1 mg/1 BAP (L3D1B1). Other conditions were as described by J~me et al. (1991a). The cultures were incubated in the dark at 26°C for 5 to 15 d (scutella) or 2 to 4 weeks (anthers).
138 Feeder cells. Microspore-derived aggregates were used as feeder cells for the culture of protoplasts derived from scutella. Microspores were isolated according to Mordhorst and L6rz (1993) and cultivated in liquid L3 medium with 1.5 mg/1 2,4-D and 0.5 rag/1 BAP (L3D1.5B0.5). The osmolality of the feeder cell medium was adjusted to 550 mosm.kg"1with mannitol. As nurse cells, 3 to 5 week-old microspore-derived calli were used. For anther culture-derived protoplasts, suspension cells of the line D2-1 derived from embryo culture of H. murinum were used. The cell suspensions were cultured according to Lazzeri et al. (1991) and used as nurse cells 1 to 3 days after subculture. Protoplast isolation, culture and regeneration. Scntella: Protoplasts were isolated according to Lazzeri et al. (1991). Primary callus developing after 5 to 15 days of culture was used for protoplast isolation (Fig. la). Twenty scntella were incubated in 20 ml enzyme solution containing 1% cellulase Onozuka RS and 0.05% pectolyase Y23 in LW solution. The osmolality was adjusted to 720 mosm.kg"x with mannitol. After 2 to 3 hours of incubation in the dark, the protoplasts were filtered through 100 ~tm, 50 ~tm and 22 ~tm nylon sieves and washed twice with LW solution. The protoplasts were embedded at a density of 106/ml in L1 medium containing 2 mg/l 2,4-D solidified with 2% Sea-Plaque agarose (FMC®). The osmolality of the medium was adjusted with mannitol to 720 mosm.kg"1. One-ml aliquots of the protoplast-containing suspension were plated on MillicellxM-CM inserts (Millipore). The inserts were placed in 6 cm petri dishes (TC-quality, Greiner). The petridishes contained 3 to 5 ml feeder cell medium with 150 mg nurse cells (Fig. Ic). Cultures were incubated at 26°C on a rotary shaker at 30 rpm in the dark. After two weeks, the feeder cells were removed and the feeder cell medium exchanged. At this time, the plating efficiency was determined. After four weeks of culture agarose which contained visible colonies was transferred to L3 induction medium supplemented with 0.5 mg/1 2.4-D and 1.5 rag/1 BAP (L3D0.5B1.5) and solidified with 0.4% agarose (Sigma Chemicals) to induce embryogenesis. Embryogenic structures were subcultured every two weeks on L3D0.5B1.5 medium until shoots developed. Green shoots were transferred onto hormone-free L3 medium for rooting, subsequently potted in soil and grown to maturity in the greenhouse.
Anthers: Three to four week-old primary callus (Fig. 2a) from 3 to 6 spiklets was incubated in 20 ml enzyme solution containing 2% cellulase RS, 0.05 % pectolyase Y23, 0.1% CaCI2 x 2 I-I20 and 0.5 M mannitol (Wu and Zapata 1992). The osmolality of the solution was 650 mosm After enzyme treatment, the protoplasts
were filtered and washed as described for protoplast isolation from scutella. The purified protoplasts were embedded at a density of 1.5 x 106/ml in L1 medium at an osmolality of 650 mosm.kg"1 containing 1 mg/l 2,4-D and solidified with 1.25% Sea-Plaque agarose. The protoplast-containing agarose was cut into pieces and placed in a 6 cm petridish surrounding a MillicelP-M-CM (MiUipore) insert. Five ml of liquid protoplast medium with suspension cells (D2-1) used as feeder cells were added to the insert (Fig. 2c). Culture conditions were the same as for protoplasts derived from primary callus of scutella. After two weeks of culture, the medium was replaced with L1 medium at an osmolality of 450 mosm.kg"1, and the feeder ceils were removed. After another week, the medium was exchanged with L1 medium at an osmolality of 300 mosm.kg"1. Cells were cultured for 1 to 3 weeks in dim light until formation of cell clusters. Calli bigger than 2 nun were plated on induction medium containing 1 mg/1 2,4-D and 1 mg/1 BAP and subcultured every two weeks. Embryogenic calli were transferred to hormone-free L2 medium for shoot formation and rooting. Plants were vernalized for six weeks in a growth chamber at 4°C with 10 h light and grown to maturity in the greenhouse. Table 1 gives an overview of the two protoplast regeneration systems.
Results and discussion Protoplast isolation and culture. A large number of protoplasts was obtained from primary callus of scutella and anthers (Figs. lb and 2b). The yield of protoplasts ranged from 2 x 106 to 8 x 106 per g fresh weight. The yield increased with the duration of the preculture. From scntella, the highest number of protoplasts was isolated after a preculture period of 10 to 15 days. Longer periods (>15 d) resulted in a lot of debris possibly due to the advanced development of the primary callus. The scutella-derived protoplasts contained more starch compared to the anther-derived protoplasts. First divisions occured after 3 to 5 days in culture (Figs. ld and 2d). Two weeks after protoplast isolation the plating efficiency was determined. Colonies were counted under the microscope. The microcalli were compact and consisted of small and cytoplasm-rich cells. The efficiency ranged from 0.2% to 0.7% for scutella-derived cultures and from 0.1% to 0.2% for anther-derived cultures. Scutella-derived protoplasts failed to divide without feeder cells. For rice (Biswas 1994, Jain et al. 1995) and barley (Kihara and Funatsuki 1995) protoplasts of non-embryogenic cell suspensions could be used as feeder. In our experiments, non-embryogenic suspension cells could only be used as nurse cells for anther-derived protoplasts, whereas the plating
139 Table 1:
Schematic protocol and time schedule of protoplast regeneration systems from scutella and anthers of barley Anthers Winter type cv. 'lgri'
Scutella Spring type cv. 'Dissa' Scutella
Preculture scutella on L2D1 in the dark at 26°C
Anthers I
5- 15 days
] Isolate protoplasts by incubation of primary callus in 1% cellulase RS and 0.075% pectolyase Y23 at 720 mosm. kg 1
2-4 weeks
Primary callus 2-4 hours
]
Embed lx106 protoplasts in lml L1D1 at 720 mosm.kg 4, solidified with 2% SP-Agarose in Millicell~--CM inserts; add microspore derived aggregates in 3 ml feeder cell medium, L3D1.5B0.5, 550 mosm.kg 4
Isolate protoplasts by incubation of primary callus in 2% cellulase RS and 0.05% pectolyase Y23 at 650 mosm.kg 4
2-4 hours
Protoplasts
]
2 weeks
2 weeks
Microcalli 1 mm 2 weeks
]
Remove feeder cells and replace feeder medium 1-3 weeks
Microcalli >_2 mm
Exchange medium at 450 mosm.kg 1 with medium at 300 mosm. kg -1
] Transfer to induction medium L3D1 B1; subculture every two weeks
4-6 weeks
Shoots Transfer to hormone-free L3 medium for rooting
2-4 weeks
[ Transfer to greenhouse
[
] Transfer to hormone-free L2 medium for rooting
2-4 weeks
Plantlets 3 months
Embed 1.5x10 e protoplasts in lml L1D1, at 650 mosm.kg 1, solidified with 1.2% SeaPlaque agarose in a petri dish; cut agarose into pieces; add suspension cells (D2-1) to a Millicellr~+-CM insert and 5 ml suspension medium Remove feeder cells and replace feeder medium with medium at 450 mosm.kg 4
1 week
Transfer to induction medium L3D0.5B1; subculture every two weeks
Pretreat spikelets for 14-21 days in the dark at 4°C. Preculture anthers on L2D1Blin the dark at 26°C
] Transfer to greenhouse *including vemalisation
6 months* Fertile plants
]
140
Fig.l: Plant regeneration of barley protoplasts (cv. "Dissa') derived from primary callus of scutella. A) Primary callus twelve days after plating scutella. B) Freshly isolated protoplasts. C) Protoplast-derived cell colonies formed from protoplasts one week after embedding. D) Visible colonies on induction medium four weeks after protoplast isolation. E) Regeneration of protoplast-derived plantlets on hormone-free medium. F) Maturing spikes from fertile plants regenerated from scutella-derived protoplasts.
Fig. 2: Plant regeneration of barley protoplasts (cv. "Igri') derived from primary callus of anthers. A) Primary callus four weeks after plating anthers. B) Freshly isolated protoplasts. C) Protoplast-derived cell colony formed from a protoplast one week after embedding. D) Visible colonies four weeks after protoplast isolation. E) Regeneration of green plantlets. F) Fertile plants regenerated from anther-derived protoplasts.
141
efficiency was strongly reduced (
Plant Cell Reports (1996) 16:137-141
© Springer-Verlag1996
Primary callus as source of totipotent barley (Hordeum vulgare L.) protoplasts Annette Sti~ldt *~ Xiao-Hui Wang *' **, and Horst Ltirz Zentrum ftir Angewandte Molekularbiologie der Pflanzen (AMP II), Institut fiir Allgemeine Botanik, Universit/it Hamburg, Ohnhorststr. 18, D-22609 Hamburg, Germany * These authors have contributed equally to the work presented ** Present address: Department of Biology, Massachusetts Institute of Technology, Building 68-617, 77 Massachusetts Avenue, Cambridge, MA 02139, USA Received 15 May 1996/Revised version received 24 May 1996 - Communicated by K. Glimelius
Summary Primary callus of barley (Hordeum vulgare L.) derived from scutella (cv. 'Dissa') and anthers (cv. 'Igri') was used for protoplast isolation and plant regeneration. The protoplasts were embedded in agarose and cultured with nurse cells. The plating efficiency varied from 0,1% to 0.7%. Shoots regenerated from the developing callus. Plantlets were transferred to soil and cultivated in the greenhouse three to five months after protoplast isolation. All plants were normal in morphology, and most of them flowered and set seeds. Key words: Protoplasts - Hordeum vulgare L. - nurse culture plant regeneration - primary callus
Abbreviations: 2,4-D: 2,4-dichlorophenoxyacetic acid, BAP: 6-benylaminopurine
(Jhhne et al 1991 a,b, Funatsuki et al, 1992, Golds et al. 1994, Kihara and Funatsuki 1995). This indicates that plant regeneration from barley protoplasts still remains a problem. As an alternative, it has been suggested recently to use primary callus as a source for protoplast isolation. In the case of barley, primary callus can be produced from immature embryos (Kihara and Funatsuld 1995), microspores (Salmenkallio-Martilla and Kauppinen 1995) or anthers. The obvious advantage of this material lies in the shortened tissue culture period and, subsequently, in a reduced risk of somaclonal variation. We present here two rapid regeneration systems for protoplasts from primary callus derived from either young scutella or anthers of barley.
Material and methods Plant material. The spring type barley cultivar 'Dissa'
Introduction Protoplasts represent a useful system for plant genetic manipulations such as somatic hybridisation, cytoplasmic recombination or direct gene transfer. For this, an efficient plant regeneration system from isolated protoplasts is a prerequisite. The major source for protoplasts of cereal crops are embryogenic cell suspensions (reviews: Vasil 1992, Krautwig and L6rz 1995). The establishment of suspension cultures producing a high yield of viable protoplasts is very laborious and strongly genotypedependent. Additional problems arise during the long period of culture with a decrease in morphogenic capacity (J~me et al. 1991a,b) and the occurrence of somaclonal variation (Karp et al. 1987, Wang et al, 1992). Up to now, there are only few reports on efficient and reliable regeneration of fertile barley plants from suspension culture-derived protoplasts Correspondence to: H. L6rz
was used for the initiation of primary callus from immature embryos. For callus induction from anthers the winter-type 'Igri' was used. Barley plants of the cultivar 'Gimpel' were grown for the isolation of microspores which were added as nurse cells. Induction o f primary callus. Immature embryos were
harvested at a length of 0.5 to 1.0 mm. The embryo axes were removed and the scutella cultured upside down on L2 medium (Lazzeri et al. 1991) supplemented with 1 mg/1 2,4-D. Spiklets containing pollen at the mid-uninucleate stage were pretreated at 4°C in the dark for 14 to 21 days. The anthers were cultured on L3 medium (Lazzeri et al. 1991) supplemented with 1 mg/1 2,4-D and 1 mg/1 BAP (L3D1B1). Other conditions were as described by J~me et al. (1991a). The cultures were incubated in the dark at 26°C for 5 to 15 d (scutella) or 2 to 4 weeks (anthers).
138 Feeder cells. Microspore-derived aggregates were used as feeder cells for the culture of protoplasts derived from scutella. Microspores were isolated according to Mordhorst and L6rz (1993) and cultivated in liquid L3 medium with 1.5 mg/1 2,4-D and 0.5 rag/1 BAP (L3D1.5B0.5). The osmolality of the feeder cell medium was adjusted to 550 mosm.kg"1with mannitol. As nurse cells, 3 to 5 week-old microspore-derived calli were used. For anther culture-derived protoplasts, suspension cells of the line D2-1 derived from embryo culture of H. murinum were used. The cell suspensions were cultured according to Lazzeri et al. (1991) and used as nurse cells 1 to 3 days after subculture. Protoplast isolation, culture and regeneration. Scntella: Protoplasts were isolated according to Lazzeri et al. (1991). Primary callus developing after 5 to 15 days of culture was used for protoplast isolation (Fig. la). Twenty scntella were incubated in 20 ml enzyme solution containing 1% cellulase Onozuka RS and 0.05% pectolyase Y23 in LW solution. The osmolality was adjusted to 720 mosm.kg"x with mannitol. After 2 to 3 hours of incubation in the dark, the protoplasts were filtered through 100 ~tm, 50 ~tm and 22 ~tm nylon sieves and washed twice with LW solution. The protoplasts were embedded at a density of 106/ml in L1 medium containing 2 mg/l 2,4-D solidified with 2% Sea-Plaque agarose (FMC®). The osmolality of the medium was adjusted with mannitol to 720 mosm.kg"1. One-ml aliquots of the protoplast-containing suspension were plated on MillicellxM-CM inserts (Millipore). The inserts were placed in 6 cm petri dishes (TC-quality, Greiner). The petridishes contained 3 to 5 ml feeder cell medium with 150 mg nurse cells (Fig. Ic). Cultures were incubated at 26°C on a rotary shaker at 30 rpm in the dark. After two weeks, the feeder cells were removed and the feeder cell medium exchanged. At this time, the plating efficiency was determined. After four weeks of culture agarose which contained visible colonies was transferred to L3 induction medium supplemented with 0.5 mg/1 2.4-D and 1.5 rag/1 BAP (L3D0.5B1.5) and solidified with 0.4% agarose (Sigma Chemicals) to induce embryogenesis. Embryogenic structures were subcultured every two weeks on L3D0.5B1.5 medium until shoots developed. Green shoots were transferred onto hormone-free L3 medium for rooting, subsequently potted in soil and grown to maturity in the greenhouse.
Anthers: Three to four week-old primary callus (Fig. 2a) from 3 to 6 spiklets was incubated in 20 ml enzyme solution containing 2% cellulase RS, 0.05 % pectolyase Y23, 0.1% CaCI2 x 2 I-I20 and 0.5 M mannitol (Wu and Zapata 1992). The osmolality of the solution was 650 mosm After enzyme treatment, the protoplasts
were filtered and washed as described for protoplast isolation from scutella. The purified protoplasts were embedded at a density of 1.5 x 106/ml in L1 medium at an osmolality of 650 mosm.kg"1 containing 1 mg/l 2,4-D and solidified with 1.25% Sea-Plaque agarose. The protoplast-containing agarose was cut into pieces and placed in a 6 cm petridish surrounding a MillicelP-M-CM (MiUipore) insert. Five ml of liquid protoplast medium with suspension cells (D2-1) used as feeder cells were added to the insert (Fig. 2c). Culture conditions were the same as for protoplasts derived from primary callus of scutella. After two weeks of culture, the medium was replaced with L1 medium at an osmolality of 450 mosm.kg"1, and the feeder ceils were removed. After another week, the medium was exchanged with L1 medium at an osmolality of 300 mosm.kg"1. Cells were cultured for 1 to 3 weeks in dim light until formation of cell clusters. Calli bigger than 2 nun were plated on induction medium containing 1 mg/1 2,4-D and 1 mg/1 BAP and subcultured every two weeks. Embryogenic calli were transferred to hormone-free L2 medium for shoot formation and rooting. Plants were vernalized for six weeks in a growth chamber at 4°C with 10 h light and grown to maturity in the greenhouse. Table 1 gives an overview of the two protoplast regeneration systems.
Results and discussion Protoplast isolation and culture. A large number of protoplasts was obtained from primary callus of scutella and anthers (Figs. lb and 2b). The yield of protoplasts ranged from 2 x 106 to 8 x 106 per g fresh weight. The yield increased with the duration of the preculture. From scntella, the highest number of protoplasts was isolated after a preculture period of 10 to 15 days. Longer periods (>15 d) resulted in a lot of debris possibly due to the advanced development of the primary callus. The scutella-derived protoplasts contained more starch compared to the anther-derived protoplasts. First divisions occured after 3 to 5 days in culture (Figs. ld and 2d). Two weeks after protoplast isolation the plating efficiency was determined. Colonies were counted under the microscope. The microcalli were compact and consisted of small and cytoplasm-rich cells. The efficiency ranged from 0.2% to 0.7% for scutella-derived cultures and from 0.1% to 0.2% for anther-derived cultures. Scutella-derived protoplasts failed to divide without feeder cells. For rice (Biswas 1994, Jain et al. 1995) and barley (Kihara and Funatsuki 1995) protoplasts of non-embryogenic cell suspensions could be used as feeder. In our experiments, non-embryogenic suspension cells could only be used as nurse cells for anther-derived protoplasts, whereas the plating
139 Table 1:
Schematic protocol and time schedule of protoplast regeneration systems from scutella and anthers of barley Anthers Winter type cv. 'lgri'
Scutella Spring type cv. 'Dissa' Scutella
Preculture scutella on L2D1 in the dark at 26°C
Anthers I
5- 15 days
] Isolate protoplasts by incubation of primary callus in 1% cellulase RS and 0.075% pectolyase Y23 at 720 mosm. kg 1
2-4 weeks
Primary callus 2-4 hours
]
Embed lx106 protoplasts in lml L1D1 at 720 mosm.kg 4, solidified with 2% SP-Agarose in Millicell~--CM inserts; add microspore derived aggregates in 3 ml feeder cell medium, L3D1.5B0.5, 550 mosm.kg 4
Isolate protoplasts by incubation of primary callus in 2% cellulase RS and 0.05% pectolyase Y23 at 650 mosm.kg 4
2-4 hours
Protoplasts
]
2 weeks
2 weeks
Microcalli 1 mm 2 weeks
]
Remove feeder cells and replace feeder medium 1-3 weeks
Microcalli >_2 mm
Exchange medium at 450 mosm.kg 1 with medium at 300 mosm. kg -1
] Transfer to induction medium L3D1 B1; subculture every two weeks
4-6 weeks
Shoots Transfer to hormone-free L3 medium for rooting
2-4 weeks
[ Transfer to greenhouse
[
] Transfer to hormone-free L2 medium for rooting
2-4 weeks
Plantlets 3 months
Embed 1.5x10 e protoplasts in lml L1D1, at 650 mosm.kg 1, solidified with 1.2% SeaPlaque agarose in a petri dish; cut agarose into pieces; add suspension cells (D2-1) to a Millicellr~+-CM insert and 5 ml suspension medium Remove feeder cells and replace feeder medium with medium at 450 mosm.kg 4
1 week
Transfer to induction medium L3D0.5B1; subculture every two weeks
Pretreat spikelets for 14-21 days in the dark at 4°C. Preculture anthers on L2D1Blin the dark at 26°C
] Transfer to greenhouse *including vemalisation
6 months* Fertile plants
]
140
Fig.l: Plant regeneration of barley protoplasts (cv. "Dissa') derived from primary callus of scutella. A) Primary callus twelve days after plating scutella. B) Freshly isolated protoplasts. C) Protoplast-derived cell colonies formed from protoplasts one week after embedding. D) Visible colonies on induction medium four weeks after protoplast isolation. E) Regeneration of protoplast-derived plantlets on hormone-free medium. F) Maturing spikes from fertile plants regenerated from scutella-derived protoplasts.
Fig. 2: Plant regeneration of barley protoplasts (cv. "Igri') derived from primary callus of anthers. A) Primary callus four weeks after plating anthers. B) Freshly isolated protoplasts. C) Protoplast-derived cell colony formed from a protoplast one week after embedding. D) Visible colonies four weeks after protoplast isolation. E) Regeneration of green plantlets. F) Fertile plants regenerated from anther-derived protoplasts.
141
efficiency was strongly reduced (
