Plant Cell Reports
Plant Cell Reports (1991) 10:277-281
9 Springer-Verlag1991
Plant regeneration from protoplasts of Panax ginseng (C. A. Meyer) through somatic embryogenesis Sarita Arya 1,3, Jang R. Liu 2, and Tage Eriksson 1 Department of Physiological Botany, University of Uppsala, Box 540, S-751 21 Uppsala, Sweden z Genetic Engineering Research Institute, KIST, Box 17, Taejon, Korea 3 Institute of Arid Zone Forestry Research, Jodhpur-342001, India Received March 1, 1991/Revised version received May 2, 1991 - Communicated by H. Lrrz
SUMMARY
Protoplasts of Panax ~insen~ were isolated from embryos obtained from the 4-year old embryogenic cell line KCTC PCL 49031 which was derived from a zygotic embryo. High protoplast yields of 22-25 xl06 protoplast / g tissue were obtained following 5-6 h digestion with 2% CeUulysin, 1% Pectinase and 1% Macerasae in half strength Murashige and Skoog's medium containing 12% mannitol. A plating density of lxl05 protoplasts/ml was found optimal for protoplast culture. An initial division frequency of 10% was obtained in an agarosegelled defined medium. Myo-inositol (6%) was found to be the most suitable osmoticum. Somatic embryos were formed from protoplast derived embryogenic callus,which regenerated into plantlets. ABBREVIATIONS: NAA, naphthaleneacetic acid ; 2,4-D, 2,4-dichlorophenoxyacetic acid ; Kn, kinetin ; BA, benzyladenine ; GA 3, gibberellic acid ; MS, Murashige and Skoog medium.
In ginseng, Butenko et al. (1968) first induced somatic embryos from root callus. Hem and Lee (1974) induced organized structures from callus of ginseng cotyledon explants and Chang and Hsing (1980) showed in vitro flowering from plantlets regenerated from mature root callus. Lee et al. (1990) induced plantlets and flowering from callus derived from zygotic embryos. At present no information is available on protoplast isolation and culture of ginseng. We describe for the first time an efficient procedure for isolation,culture and regeneration of protoplasts into plantlets of Panax ginsen~ through somatic embryogenesis. A 4-year old embryogenic cell line of Panax ginsen~ capable of regeneration and flowering was used. The aims of the experiments were twofold: 1) to establish the conditions where by a sufficient number of protoplasts could be isolated routinely for their use in direct gene transfer, and 2) to optimize the conditions for protoplast division, growth of callus and regeneration of plants. MATERIALS AND METHODS
INTRODUCTION
Establishment of embrvo~enic callus cultures
P a n a x g i n s e n g C.A.Meyer (Araliaceae) is an economically important medicinal herb used as a vitalizing and stimulating agent. Some of its pharmacologically active substances are triterpene saponins and ginsenosides (Shibata et al. 1985). The root has been used as a drug since ancient times and is expensive due to a long and complicated cultivation (5-7 years). A three year juvenile period has hampered conventional breeding programs for its improvement, since a long time is needed for genetic analysis. An efficient protoplast to plant system would make ginseng amenable to direct gene transfer and for production of promising asymmetric somatic hybrids, e.g. between ginseng and carrot with ginsenosides properties. Such asymmetric hybrids may be easier to grow than the normal ginseng plant.
An embryogenic culture line KCTC PCL 49031 derived from a zygotic embryo of Panax ginsen~ was used. Dehusked seeds of ginseng which had been stored at 4~ C in the dark were surface sterilized with 70% ethanol for 5 min and subsequently in 50% clorox solution for 20 rain. The seeds were rinsed three times with sterile double distilled water before zygotic embryos were dissected out and placed on agarose solidified (0.7%) MS (Murashige and Skoog 1962) medium supplemented with 1.0 mg/l 2,4-D, 0.01 mg/l Kn and 30 g sucrose/l. The medium was adjusted to pH 5.8 before autoclaving. The cultures were kept in dark at 25 ~ C for 10 weeks. Callus formation was observed after 4 weeks of culture. After 6 weeks callus gave rise to somatic embryos from the entire surface of the callus.The callus was subcultured at 4 week intervals and maintained under 16 h photoperiod (30 lamol m-2s-1, Osram 36W/30 warm white) at 20~ C.
Offprint requests to: S. Arya
278
Isolation of Protoolast$ Somatic embryos isolated from embryogenic callus were used as protoplast source. Embryos (250 mg) were preplasmolysed for 1 h in a solution containing 0.3 M sorbitol and 0.5 M CaCI2 and were incubated in 10 ml of enzyme solution in a Petridish (9 cm Falcon). Embryos were mechanically pressed with fine forceps to loosen the cells before incubation. The enzyme mixture consisted of 2% Cellulysin (Calbiochem), 1% Pectinase (Sigma), 1% Macerase (Calbiochem), 0.5% potassium dextran sulphate and 12% mannitol in half strength MS medium at pH 5.6. The enzyme solution used was filter sterilized. The cultures were kept on a gyratory shaker for 5-6 h in the dark at 25 ~ C, initially 1 h at 50 rpm and then at 40 rpm. The resulting suspension was passed through 100 lain and 50 ~m mesh respectively to separate protoplasts from undigested debris. Protoplasts were washed in an equal volume of CPW 16 salts (Banks and Evans 1976) and centrifuged at 70 x g for 5 min in a swing bucket rotor. The pellet was washed twice in W5 medium (Menczel et al. 1981) and once in t h e culture medium . The protoplasts were resuspended finally in culture medium consisting of half strength M8P (Glimelius et a1.1986) with myo-inositol (0.33M), 1% glucose, 1% sorbitol, 0.5% galactose, 1.0 mg/1 2,4-D, 0.5 mg/1 BA and 0.5 mg/l Kn at pH 5.8. The medium used was filter sterilized. The cell wall digestion was checked by staining with Calcofluor white as described by Galbraith (1981) and viability was tested by Trypan blue exclusion. The protoplast ~ields were estimated by using a haemocytometer on an inverted microscope and protoplasts were diluted to the desired plating density.
Different osmotica viz. glucose, sucrose, mannitol, sorbitol and myo-inositol were used in agarose beads to test the initial plating efficiency and colony formation (Table 2) at a density of 105 protoplasts / ml. Half of the protoplast medium was replaced by fresh culture medium at a one week interval with a lower osmolarity.
Somatic embryogenesis and regeneration The calli with diameters of 1-2 mm were transferred to agarose solidified PCM III medium (Table 3) at pH 5.8, 16 h photoperiod (30 lamol m'2s "1, Osram 36W/30 warm white) where calli grew and formed somatic embryos. Mature somatic embryos were placed on regeneration medium (PCM IV) for plant formation (Table 3). RESULT AND DISCUSSION
Isolation and culture of t~rotoolasts Somatic embryos isolated from embryogenic callus were found to be the most suitable source for protoplast isolation of ginseng (Fig 1, 2). Such embryos gave clean preparations of intact protoplasts rich in cytoplasm (Fig 3). Several other workers also found embryogenic cell lines as better sources of protoplasts; in wheat (Vasil et al. 1990), corn (Rhodes et al. 1988), cotton (Wang 1989), and black spruce (Tautorus et al. 1990). The isolation procedure described routinely yielded approximately 22-25 xl06 protoplasts per gram somatic embryos of Panax ginseng. Protoplast release started within 3 h o t enzymatic digestion but 5-6 h incubation was necessary for complete digestion of the cell wall.
Protot~last Culture Three protoplast culture methods were evaluated initially : in liquid culture medium (2.5 ml) in 5 cm Petri dishes; embedded in 0.6% agarose (Seaplaque) in bead culture in which 150 lxl droplets were formed in a Petri dish, or in 2 mm thick agarose plates. After 24 h, 3 ml liquid culture medium was added to the agarose bead and agarose plates. Protoplast density for these experiments was 7.5 x 104 -1 x 105/ml. All cultures were incubated in the dark at 250 C. To determine the influence of plating density on plating efficiency and colony formation, protoplasts were tested at densities of 2.5 x 104 - 2.0 x 105 /ml (Table 1) using the agarose bead culture system.The plating efficiency was determined after 10 days of culture and referred to the number of protoplasts divided once or twice. Plating efficiencies were estimated as follows;
Optimal protoplast culture density was found to be lxl05 protoplast/ml (Table 1) as also reported by many workers for other protoplast system (Barsby et al. 1986, Miura and Mamoru 1986, Gleddie et al. 1989). Embedding of protoplasts in agarose gave a higher plating efficiency than liquid culture. In liquid culture most of the protoplasts adhered to each other and died. A more complex medium i.e. M8P as used for Brassica (Glimelius et al. 1986) was found to be most suitable for protoplast divisions and colony formation in ginseng.
Protoplast density/ml
Plating efficiency(%)
Plating efficiency =
25,000
Plant Cell Reports (1991) 10:277-281
9 Springer-Verlag1991
Plant regeneration from protoplasts of Panax ginseng (C. A. Meyer) through somatic embryogenesis Sarita Arya 1,3, Jang R. Liu 2, and Tage Eriksson 1 Department of Physiological Botany, University of Uppsala, Box 540, S-751 21 Uppsala, Sweden z Genetic Engineering Research Institute, KIST, Box 17, Taejon, Korea 3 Institute of Arid Zone Forestry Research, Jodhpur-342001, India Received March 1, 1991/Revised version received May 2, 1991 - Communicated by H. Lrrz
SUMMARY
Protoplasts of Panax ~insen~ were isolated from embryos obtained from the 4-year old embryogenic cell line KCTC PCL 49031 which was derived from a zygotic embryo. High protoplast yields of 22-25 xl06 protoplast / g tissue were obtained following 5-6 h digestion with 2% CeUulysin, 1% Pectinase and 1% Macerasae in half strength Murashige and Skoog's medium containing 12% mannitol. A plating density of lxl05 protoplasts/ml was found optimal for protoplast culture. An initial division frequency of 10% was obtained in an agarosegelled defined medium. Myo-inositol (6%) was found to be the most suitable osmoticum. Somatic embryos were formed from protoplast derived embryogenic callus,which regenerated into plantlets. ABBREVIATIONS: NAA, naphthaleneacetic acid ; 2,4-D, 2,4-dichlorophenoxyacetic acid ; Kn, kinetin ; BA, benzyladenine ; GA 3, gibberellic acid ; MS, Murashige and Skoog medium.
In ginseng, Butenko et al. (1968) first induced somatic embryos from root callus. Hem and Lee (1974) induced organized structures from callus of ginseng cotyledon explants and Chang and Hsing (1980) showed in vitro flowering from plantlets regenerated from mature root callus. Lee et al. (1990) induced plantlets and flowering from callus derived from zygotic embryos. At present no information is available on protoplast isolation and culture of ginseng. We describe for the first time an efficient procedure for isolation,culture and regeneration of protoplasts into plantlets of Panax ginsen~ through somatic embryogenesis. A 4-year old embryogenic cell line of Panax ginsen~ capable of regeneration and flowering was used. The aims of the experiments were twofold: 1) to establish the conditions where by a sufficient number of protoplasts could be isolated routinely for their use in direct gene transfer, and 2) to optimize the conditions for protoplast division, growth of callus and regeneration of plants. MATERIALS AND METHODS
INTRODUCTION
Establishment of embrvo~enic callus cultures
P a n a x g i n s e n g C.A.Meyer (Araliaceae) is an economically important medicinal herb used as a vitalizing and stimulating agent. Some of its pharmacologically active substances are triterpene saponins and ginsenosides (Shibata et al. 1985). The root has been used as a drug since ancient times and is expensive due to a long and complicated cultivation (5-7 years). A three year juvenile period has hampered conventional breeding programs for its improvement, since a long time is needed for genetic analysis. An efficient protoplast to plant system would make ginseng amenable to direct gene transfer and for production of promising asymmetric somatic hybrids, e.g. between ginseng and carrot with ginsenosides properties. Such asymmetric hybrids may be easier to grow than the normal ginseng plant.
An embryogenic culture line KCTC PCL 49031 derived from a zygotic embryo of Panax ginsen~ was used. Dehusked seeds of ginseng which had been stored at 4~ C in the dark were surface sterilized with 70% ethanol for 5 min and subsequently in 50% clorox solution for 20 rain. The seeds were rinsed three times with sterile double distilled water before zygotic embryos were dissected out and placed on agarose solidified (0.7%) MS (Murashige and Skoog 1962) medium supplemented with 1.0 mg/l 2,4-D, 0.01 mg/l Kn and 30 g sucrose/l. The medium was adjusted to pH 5.8 before autoclaving. The cultures were kept in dark at 25 ~ C for 10 weeks. Callus formation was observed after 4 weeks of culture. After 6 weeks callus gave rise to somatic embryos from the entire surface of the callus.The callus was subcultured at 4 week intervals and maintained under 16 h photoperiod (30 lamol m-2s-1, Osram 36W/30 warm white) at 20~ C.
Offprint requests to: S. Arya
278
Isolation of Protoolast$ Somatic embryos isolated from embryogenic callus were used as protoplast source. Embryos (250 mg) were preplasmolysed for 1 h in a solution containing 0.3 M sorbitol and 0.5 M CaCI2 and were incubated in 10 ml of enzyme solution in a Petridish (9 cm Falcon). Embryos were mechanically pressed with fine forceps to loosen the cells before incubation. The enzyme mixture consisted of 2% Cellulysin (Calbiochem), 1% Pectinase (Sigma), 1% Macerase (Calbiochem), 0.5% potassium dextran sulphate and 12% mannitol in half strength MS medium at pH 5.6. The enzyme solution used was filter sterilized. The cultures were kept on a gyratory shaker for 5-6 h in the dark at 25 ~ C, initially 1 h at 50 rpm and then at 40 rpm. The resulting suspension was passed through 100 lain and 50 ~m mesh respectively to separate protoplasts from undigested debris. Protoplasts were washed in an equal volume of CPW 16 salts (Banks and Evans 1976) and centrifuged at 70 x g for 5 min in a swing bucket rotor. The pellet was washed twice in W5 medium (Menczel et al. 1981) and once in t h e culture medium . The protoplasts were resuspended finally in culture medium consisting of half strength M8P (Glimelius et a1.1986) with myo-inositol (0.33M), 1% glucose, 1% sorbitol, 0.5% galactose, 1.0 mg/1 2,4-D, 0.5 mg/1 BA and 0.5 mg/l Kn at pH 5.8. The medium used was filter sterilized. The cell wall digestion was checked by staining with Calcofluor white as described by Galbraith (1981) and viability was tested by Trypan blue exclusion. The protoplast ~ields were estimated by using a haemocytometer on an inverted microscope and protoplasts were diluted to the desired plating density.
Different osmotica viz. glucose, sucrose, mannitol, sorbitol and myo-inositol were used in agarose beads to test the initial plating efficiency and colony formation (Table 2) at a density of 105 protoplasts / ml. Half of the protoplast medium was replaced by fresh culture medium at a one week interval with a lower osmolarity.
Somatic embryogenesis and regeneration The calli with diameters of 1-2 mm were transferred to agarose solidified PCM III medium (Table 3) at pH 5.8, 16 h photoperiod (30 lamol m'2s "1, Osram 36W/30 warm white) where calli grew and formed somatic embryos. Mature somatic embryos were placed on regeneration medium (PCM IV) for plant formation (Table 3). RESULT AND DISCUSSION
Isolation and culture of t~rotoolasts Somatic embryos isolated from embryogenic callus were found to be the most suitable source for protoplast isolation of ginseng (Fig 1, 2). Such embryos gave clean preparations of intact protoplasts rich in cytoplasm (Fig 3). Several other workers also found embryogenic cell lines as better sources of protoplasts; in wheat (Vasil et al. 1990), corn (Rhodes et al. 1988), cotton (Wang 1989), and black spruce (Tautorus et al. 1990). The isolation procedure described routinely yielded approximately 22-25 xl06 protoplasts per gram somatic embryos of Panax ginseng. Protoplast release started within 3 h o t enzymatic digestion but 5-6 h incubation was necessary for complete digestion of the cell wall.
Protot~last Culture Three protoplast culture methods were evaluated initially : in liquid culture medium (2.5 ml) in 5 cm Petri dishes; embedded in 0.6% agarose (Seaplaque) in bead culture in which 150 lxl droplets were formed in a Petri dish, or in 2 mm thick agarose plates. After 24 h, 3 ml liquid culture medium was added to the agarose bead and agarose plates. Protoplast density for these experiments was 7.5 x 104 -1 x 105/ml. All cultures were incubated in the dark at 250 C. To determine the influence of plating density on plating efficiency and colony formation, protoplasts were tested at densities of 2.5 x 104 - 2.0 x 105 /ml (Table 1) using the agarose bead culture system.The plating efficiency was determined after 10 days of culture and referred to the number of protoplasts divided once or twice. Plating efficiencies were estimated as follows;
Optimal protoplast culture density was found to be lxl05 protoplast/ml (Table 1) as also reported by many workers for other protoplast system (Barsby et al. 1986, Miura and Mamoru 1986, Gleddie et al. 1989). Embedding of protoplasts in agarose gave a higher plating efficiency than liquid culture. In liquid culture most of the protoplasts adhered to each other and died. A more complex medium i.e. M8P as used for Brassica (Glimelius et al. 1986) was found to be most suitable for protoplast divisions and colony formation in ginseng.
Protoplast density/ml
Plating efficiency(%)
Plating efficiency =
25,000
