Plant Cell Reports
Plant Cell Reports (1996) 16:88-91
© Springer-Verlag 1996
Plant regeneration from protoplasts isolated from callus of Gentiana crassicaulis Yu-ling Meng, Yun-peng Gao, and Jing-fen Jia Biology Department, Lanzhou University, Lanzhou, Gansu 730000, P. R. China Received 19 July 1995/Revisedversion received 21 December 1995 - Communicated by I. K. Vasil
Abstract Fast growing calli induced from hypocotyl segments of Gentiana crassicaulis were used for preparation of protoplasts. High yields of viable protoplasts were produced in an enzyme solution containing 1-2% cellulase, 1% pectinase, and 0.5% Hemicellulase. Protoplasts were cultured in KM8P medium containing 1 rag/1 2,4-D, 0.5 mg/l 6BA, 500 mg/l LH, 0.5 M glucose and 0.1 M mannitol by the solid-liquid dual layer culture method. First division occurred within 4-5 days of culture at a frequency of 17.8%. Sustained divisions led to callus formation. Periodically diluting the cultures with freshly prepared liquid medium containing 2% glucose was critical for colony formation. Protocolonies about 2 mm in size were transferred onto MS medium supplemented with 3 mg/1 ZT, 2 mg/1 6BA, 1 mg/l GA3, 1 mg/l NAA and 6% sucrose to obtain embryogenic calli. Plantlets were regenerated via somatic embryogenesis at high frequency on hormone-free MS Medium.
major alkaloids in Gentiana have been studied. The alkaloids exhibited significant antipsychotic (Bhattacharya et al 1974), antiinflammatory (Sadritdinov 1971), and sedative (Tulyaganov et al 1971) activity. The roots are the source of gentianine and gentianal. Traditionally, the pharmaceutical industry has largely depended on wild sources of Gentiana. G.crassicaulis is not cultivated in China. Therefore, employing plant tissue culture technique 2or its rapid clonal propagation, in vitro conservation and genetic manipulation is of great economic value. There is only one report dealing with callus formation from protoplasts of G.scabra (Zhou et al 1985). This article describes plant regeneration from protoplasts of G. crassicaulis.
Materials and Methods Callus induction
Key words: Gentiana crassicaulis Duthie ex Burk, protoplasts, callus, plant regeneration
Abbreviations 6BA: 6-benzylaminopurine, NAA: naphthaleneacetic acid, 2,4-D: 2,4-dichlorophenoxyacetic acid, ZT: zeatin, GA3: gibberellic acid, LH: lactalbumin hydrolysate, MES: 2-(N-morpholino)-ethane sulfonic acid, MS: Murashige & Skoog's medium(1962)
Introduction Gentiana crassicaulis is one of the four Commercial Radix Gentianae Macrophyllae stipulated by the Chinese Pharmacopoeia. The psychopharmacological profiles o f
Correspondence to: Jing-fen Jia
Seeds of Gentiana crassicaulis Duthie ex Burk were provided by Northwestern Plateau Biology Institute of Academia Sinica. The seeds were surface- sterilized in 0.2% Mercuric chloride(HgC12 ) for 20 min and rinsed 4 times (5 min each) in sterile distilled water. Sterilized seeds were placed on hormone-free MS medium plus 2% sucrose and 0.7% agar, pH5.8, and incubated at 25 + 2 °C under fluorescent light (800 Lux). Hypocotyls from four week old seedlings (about 2-3cm in length) were cut into 5 mm segments and cultured on MS medium plus 2 mg/1 2,4-D, 0.5 mg/1 6BA and 0.7"/0 agar to induce callus. Two weeks after culture, calli were transferred to MS medium containing 1 rag/1 2,4-D, 0.5mg/1 6BA, 500 mg/l LH and 4% sucrose, and subcultured every three weeks:
89
Protoplast isolation and culture About 1 g calli of 10-15 day old was placed in 10 ml enzyme solution containing different combinations of cellulase R-.10, macerozyme R-10 (Kinki Yakult Manufacturing Co. Ltd. Mishinomiya, Japan), pectinase (Serva Co. USA), hemicellulase (Sigma Chemical Co., St. Louis, MO,USA) (Table 1). After 810 hours incubation on a shaker (50rpm) at 25 -+ 1 °C in the dark, the released protoplasts were filtered through two stainless steel sieves (75 ~ m and 40 ~ m) and centrifuged at 500 rpm for 5 min. The pellets were resuspended in wash solution consisting of 0.18 M Calcium chloride (CaCI2) and 0.1% (w/v) MES, and layered on a volume 21% (w/v) sucrose solution. After centrifugation at 80 rpm for 10 min, the protoplasts were concentrated at the interface of the two solutions, while the debris sank to the bottom of sucrose solution. Collected protoplasts were washed three times with wash solution, and once with culture medium. Protoplasts were resuspended in KM8P medium (Kao and Michayluk 1975) supplemented with 1 mg/1 2,4-D, 0.5 mg/1 6BA, 500 mg/1 LH, 0.5 M glucose, and 0.1 M mannitol, and adjusted to a density of 1 ~ 105 protoplasts/ml. Liquid thin layer culture method and liquid-solid dual layer culture method were used for protoplast culture. The dual layer culture method involved suspending protoplasts in liquid KM8P medium and pouring 2 ml protoplast suspension into a 60~ 15mm petri dish in which 0.4% agarose containing KM8P medium were placed previously. All cultures were incubated at 25 °C in the dark. Protoplast viability was estimated by staining with 0.1% phenosafranine dissovled in 0.4 M mannitol solution. After first division occurred, the osmotic pressure of the liquid medium was gradually reduced by weekly adding supplemental medium containing 1% (w/v) glucose. The components of supplemental medium the
same as the initial culture medium, but the concentration of glucose was lowered to 1% (w/v). Plating efficiency (PE) (numbers of protocolonies visible by naked eye/ the total number of inoculated protoplasts ~ 100%) was evaluated after 30 days, The protocolonies (2 mm in diameter) were transferred to MS medium supplemented with 1 mg/1 2,4-D, 0.5 mt¢l 6BA, 500 rag/1 LH and 4% (w/v) sucrose for fimher proliferation. Plant regeneration Protoplast-derived calli were transferred onto MS medium containing 2 mg/1 6BA, 3 mg/l ZT, 1 rag/1 NAA, 1 mg/1 GA3, 500 mg/1 LH and 6% (w/v) sucrose. Three weeks later, embryogenic calli were picked up and placed on hormone-free MS medium with 1200 lux illumination at 25-+ 2°C to promote planflet development. Results and discussion
Protoplast isolation Different combinations of enzyme solution were tested for protolast isolation. As shown in Table 1, the combination of 1-2% cellulase R-10, 1% pectiase alld 0.5% hemicellulase was more suitable for efficient protoplast isolation. When the concentration of cellulase R-10 exceeded 2%, the viability of protoplasts dropped significantly. Hemicellulase was beneficial tbr protoplast isolation. Using macerozyme R-10 instead of pectinase gave lower protoplast yield and viability. Moreover, it is advantageous to reduce the incubation time of callus-enzyme mixture so that the viability of protoplasts could be improved. In our experiments, 810h was optimal.
Table T 1.h e effect, of en ~ m e combination on ~ y i e l d and ..~a~lill~* = Cellulase Pectinase Macerozyme Hemicellulase Yield of Viability of OnozukaR-10 Serve OnozukaR-10 Sigma Protoplasts protoplasts % % % % prOW % 1 1 0 0 2 x 105 90 1 1 0 0.5 1-2 ~ l0 G 90 2 1 0 1 1-2 ~ 106 85 1 0 0.5 0.5 1 ~~104 70 3 0 1 1 5 x 105 70 *Enzymes were dissolved in the solution containing KM8P inorganic components, 4.5 mM CaC12,0.5 M glucose, 0.1 M mannitol, 0.1% MES (pH5.8).
90
Figurel. A-J Plant regeneration from protoplasts of Gentiana crassicaulis callus. A: Freshly isolated protoplasts. Bar=50 la m B. First division. Bar=100 ~ m C. Second division. Bar=50 jam D.E. Clusters from protoplast-derivedcells. Bar=50 ~ m F. Protoplast-derived embryogenic callus. Bar=lmm G. Embryoids at different stages. Bar=2mm H.I. Plantlets from protoplasts via somatic embryogenesis. J. Protoplasts regenerated plants potted in greenhouse
91
Protoplast culture and callus fornmtion Freshly isolated protoplasts (Fig. IA) were cultured in KM8P medium containing 0.3 to 0.6 M glucose and 0.1 M mannitol. Glucose(0.5M) in combination with mannitol(0.1M) was the favourite osmoticum for initiating protoplast division. After 3 days in culture, most of the protoplasts increased in volume. They became elongated to oval in shape indicating new cell wall regeneration. First division occurred on the 4-5th day (Fig. 1B). Although the liquid culture method has been routinely used for initial ptotoplast culture in many species, for G.crassicaulis the solid-liquid dual layer culture method is more effective. Since protoplasts of G. crassicaulis were prone to aggregation in liquid medium, browning occurred quickly. The protoplasts failed to divide further in liquid surface. Periodically replenishing the cultures with fresh media did not delay browning. In the solidliquid dual layer cultures, first divisions were also observed on the 4-5th day. Division frequency on the 8th day reached upto 17.8% which was higher than that in liquid medium(5.8%). In the case of solid-liquid dual layer culture method, the protoplast-derived ceils divided continuously and formed protocolonies in 30 days (Fig. 1 C-E). After transfer on agarose-solidified MS medium plus 1 mg/1 2.4-D, 0.5 mg/1 6BA, 4% sucrose and 0.4% agarose, the colonies proliferated and grew into calli. The plating efficiency (PE) was between 0.2% to 0.7%. Osmotic concentration in the medium is one of the most important factors effecting the division frequency of protoplasts. The reduction of osmoticum is recommended for continued growth of protoplasts (Davey et al 1974, Gosch et al 1975). The continuous division of G. crassicauIis protoplasts could be obtained only when osmoticum was reduced gradually. Different combinations of plant growth regulators influenced the division of cultured protoplasts. 2,4-D (ling/l) in combination with 6BA(0.5mg/1) was the most suitable hormone conbination. If the level of 2,4-D and 6BA was higher than 2 mg/1, protoplast division was inhibited Plant regeneration via somatic embryogenesis. Protoplast-derived calti cultured on the differentiation medium (described in Materials and methods) turned embryogenic callus. Approximately three weeks later a yellowish and granular callus was formed on the surface of original calli (Fig. 1F). Careful selection of such embryogenic calli and their culture on hormonefree MS medium led to the formation of embryoids (Fig. 1G). Complete plantlets were obtained (Fig.H-I),
and grew in the greenhouse (Fig.lJ). Embryogenic calli have been subcultured on hormone-free MS medium for about one year, and they have maintain ed the ability to regenerate plantlets with nearly 100% differentiation frequency. But hundreds of regenerated plantlets, about 30% survived and grew normally.
Reference
I. Bhattacharya SK, Ghosal S, Chaudhuri RK, Simgh AK and Sharma PV (1974) J. Pharm.Sci. 63:13411342. 2. Davey MR, Bush E and Power JB (1974) Plant Sci. Letters 3:127-133. 3. Gosch G, Bajaj YPS and Reinert J (1975) Protoplasma 8 6 : 4 0 5 - 410. 4. Kao KN and Michayluk MR (1975) Planta 126:105110 5. Murashige T and Skoog F (1962) Physiol. Plant 15:473-493 6. Sadritdinov F (1971) In: Sultanov MB (eds) Farmakol.Alkaloidov serdechnykh. Glikozidov."Faff': Tashkent, USSR, pp146-148 7.Tulyaganov N, Danilevskii BL, Sadritdinov FS (1971) In:Sultanov MB (eds) Farmakol. Alkaloidov Serdech. Glikozidov."Fan":Tashkent, USSR.,pp148151 8. Zhou YL, Qian YQ, Cai QG, Zhang ZG and Yan X (1985) Acta Botanica Sinica 27(2): 148-150
Plant Cell Reports (1996) 16:88-91
© Springer-Verlag 1996
Plant regeneration from protoplasts isolated from callus of Gentiana crassicaulis Yu-ling Meng, Yun-peng Gao, and Jing-fen Jia Biology Department, Lanzhou University, Lanzhou, Gansu 730000, P. R. China Received 19 July 1995/Revisedversion received 21 December 1995 - Communicated by I. K. Vasil
Abstract Fast growing calli induced from hypocotyl segments of Gentiana crassicaulis were used for preparation of protoplasts. High yields of viable protoplasts were produced in an enzyme solution containing 1-2% cellulase, 1% pectinase, and 0.5% Hemicellulase. Protoplasts were cultured in KM8P medium containing 1 rag/1 2,4-D, 0.5 mg/l 6BA, 500 mg/l LH, 0.5 M glucose and 0.1 M mannitol by the solid-liquid dual layer culture method. First division occurred within 4-5 days of culture at a frequency of 17.8%. Sustained divisions led to callus formation. Periodically diluting the cultures with freshly prepared liquid medium containing 2% glucose was critical for colony formation. Protocolonies about 2 mm in size were transferred onto MS medium supplemented with 3 mg/1 ZT, 2 mg/1 6BA, 1 mg/l GA3, 1 mg/l NAA and 6% sucrose to obtain embryogenic calli. Plantlets were regenerated via somatic embryogenesis at high frequency on hormone-free MS Medium.
major alkaloids in Gentiana have been studied. The alkaloids exhibited significant antipsychotic (Bhattacharya et al 1974), antiinflammatory (Sadritdinov 1971), and sedative (Tulyaganov et al 1971) activity. The roots are the source of gentianine and gentianal. Traditionally, the pharmaceutical industry has largely depended on wild sources of Gentiana. G.crassicaulis is not cultivated in China. Therefore, employing plant tissue culture technique 2or its rapid clonal propagation, in vitro conservation and genetic manipulation is of great economic value. There is only one report dealing with callus formation from protoplasts of G.scabra (Zhou et al 1985). This article describes plant regeneration from protoplasts of G. crassicaulis.
Materials and Methods Callus induction
Key words: Gentiana crassicaulis Duthie ex Burk, protoplasts, callus, plant regeneration
Abbreviations 6BA: 6-benzylaminopurine, NAA: naphthaleneacetic acid, 2,4-D: 2,4-dichlorophenoxyacetic acid, ZT: zeatin, GA3: gibberellic acid, LH: lactalbumin hydrolysate, MES: 2-(N-morpholino)-ethane sulfonic acid, MS: Murashige & Skoog's medium(1962)
Introduction Gentiana crassicaulis is one of the four Commercial Radix Gentianae Macrophyllae stipulated by the Chinese Pharmacopoeia. The psychopharmacological profiles o f
Correspondence to: Jing-fen Jia
Seeds of Gentiana crassicaulis Duthie ex Burk were provided by Northwestern Plateau Biology Institute of Academia Sinica. The seeds were surface- sterilized in 0.2% Mercuric chloride(HgC12 ) for 20 min and rinsed 4 times (5 min each) in sterile distilled water. Sterilized seeds were placed on hormone-free MS medium plus 2% sucrose and 0.7% agar, pH5.8, and incubated at 25 + 2 °C under fluorescent light (800 Lux). Hypocotyls from four week old seedlings (about 2-3cm in length) were cut into 5 mm segments and cultured on MS medium plus 2 mg/1 2,4-D, 0.5 mg/1 6BA and 0.7"/0 agar to induce callus. Two weeks after culture, calli were transferred to MS medium containing 1 rag/1 2,4-D, 0.5mg/1 6BA, 500 mg/l LH and 4% sucrose, and subcultured every three weeks:
89
Protoplast isolation and culture About 1 g calli of 10-15 day old was placed in 10 ml enzyme solution containing different combinations of cellulase R-.10, macerozyme R-10 (Kinki Yakult Manufacturing Co. Ltd. Mishinomiya, Japan), pectinase (Serva Co. USA), hemicellulase (Sigma Chemical Co., St. Louis, MO,USA) (Table 1). After 810 hours incubation on a shaker (50rpm) at 25 -+ 1 °C in the dark, the released protoplasts were filtered through two stainless steel sieves (75 ~ m and 40 ~ m) and centrifuged at 500 rpm for 5 min. The pellets were resuspended in wash solution consisting of 0.18 M Calcium chloride (CaCI2) and 0.1% (w/v) MES, and layered on a volume 21% (w/v) sucrose solution. After centrifugation at 80 rpm for 10 min, the protoplasts were concentrated at the interface of the two solutions, while the debris sank to the bottom of sucrose solution. Collected protoplasts were washed three times with wash solution, and once with culture medium. Protoplasts were resuspended in KM8P medium (Kao and Michayluk 1975) supplemented with 1 mg/1 2,4-D, 0.5 mg/1 6BA, 500 mg/1 LH, 0.5 M glucose, and 0.1 M mannitol, and adjusted to a density of 1 ~ 105 protoplasts/ml. Liquid thin layer culture method and liquid-solid dual layer culture method were used for protoplast culture. The dual layer culture method involved suspending protoplasts in liquid KM8P medium and pouring 2 ml protoplast suspension into a 60~ 15mm petri dish in which 0.4% agarose containing KM8P medium were placed previously. All cultures were incubated at 25 °C in the dark. Protoplast viability was estimated by staining with 0.1% phenosafranine dissovled in 0.4 M mannitol solution. After first division occurred, the osmotic pressure of the liquid medium was gradually reduced by weekly adding supplemental medium containing 1% (w/v) glucose. The components of supplemental medium the
same as the initial culture medium, but the concentration of glucose was lowered to 1% (w/v). Plating efficiency (PE) (numbers of protocolonies visible by naked eye/ the total number of inoculated protoplasts ~ 100%) was evaluated after 30 days, The protocolonies (2 mm in diameter) were transferred to MS medium supplemented with 1 mg/1 2,4-D, 0.5 mt¢l 6BA, 500 rag/1 LH and 4% (w/v) sucrose for fimher proliferation. Plant regeneration Protoplast-derived calli were transferred onto MS medium containing 2 mg/1 6BA, 3 mg/l ZT, 1 rag/1 NAA, 1 mg/1 GA3, 500 mg/1 LH and 6% (w/v) sucrose. Three weeks later, embryogenic calli were picked up and placed on hormone-free MS medium with 1200 lux illumination at 25-+ 2°C to promote planflet development. Results and discussion
Protoplast isolation Different combinations of enzyme solution were tested for protolast isolation. As shown in Table 1, the combination of 1-2% cellulase R-10, 1% pectiase alld 0.5% hemicellulase was more suitable for efficient protoplast isolation. When the concentration of cellulase R-10 exceeded 2%, the viability of protoplasts dropped significantly. Hemicellulase was beneficial tbr protoplast isolation. Using macerozyme R-10 instead of pectinase gave lower protoplast yield and viability. Moreover, it is advantageous to reduce the incubation time of callus-enzyme mixture so that the viability of protoplasts could be improved. In our experiments, 810h was optimal.
Table T 1.h e effect, of en ~ m e combination on ~ y i e l d and ..~a~lill~* = Cellulase Pectinase Macerozyme Hemicellulase Yield of Viability of OnozukaR-10 Serve OnozukaR-10 Sigma Protoplasts protoplasts % % % % prOW % 1 1 0 0 2 x 105 90 1 1 0 0.5 1-2 ~ l0 G 90 2 1 0 1 1-2 ~ 106 85 1 0 0.5 0.5 1 ~~104 70 3 0 1 1 5 x 105 70 *Enzymes were dissolved in the solution containing KM8P inorganic components, 4.5 mM CaC12,0.5 M glucose, 0.1 M mannitol, 0.1% MES (pH5.8).
90
Figurel. A-J Plant regeneration from protoplasts of Gentiana crassicaulis callus. A: Freshly isolated protoplasts. Bar=50 la m B. First division. Bar=100 ~ m C. Second division. Bar=50 jam D.E. Clusters from protoplast-derivedcells. Bar=50 ~ m F. Protoplast-derived embryogenic callus. Bar=lmm G. Embryoids at different stages. Bar=2mm H.I. Plantlets from protoplasts via somatic embryogenesis. J. Protoplasts regenerated plants potted in greenhouse
91
Protoplast culture and callus fornmtion Freshly isolated protoplasts (Fig. IA) were cultured in KM8P medium containing 0.3 to 0.6 M glucose and 0.1 M mannitol. Glucose(0.5M) in combination with mannitol(0.1M) was the favourite osmoticum for initiating protoplast division. After 3 days in culture, most of the protoplasts increased in volume. They became elongated to oval in shape indicating new cell wall regeneration. First division occurred on the 4-5th day (Fig. 1B). Although the liquid culture method has been routinely used for initial ptotoplast culture in many species, for G.crassicaulis the solid-liquid dual layer culture method is more effective. Since protoplasts of G. crassicaulis were prone to aggregation in liquid medium, browning occurred quickly. The protoplasts failed to divide further in liquid surface. Periodically replenishing the cultures with fresh media did not delay browning. In the solidliquid dual layer cultures, first divisions were also observed on the 4-5th day. Division frequency on the 8th day reached upto 17.8% which was higher than that in liquid medium(5.8%). In the case of solid-liquid dual layer culture method, the protoplast-derived ceils divided continuously and formed protocolonies in 30 days (Fig. 1 C-E). After transfer on agarose-solidified MS medium plus 1 mg/1 2.4-D, 0.5 mg/1 6BA, 4% sucrose and 0.4% agarose, the colonies proliferated and grew into calli. The plating efficiency (PE) was between 0.2% to 0.7%. Osmotic concentration in the medium is one of the most important factors effecting the division frequency of protoplasts. The reduction of osmoticum is recommended for continued growth of protoplasts (Davey et al 1974, Gosch et al 1975). The continuous division of G. crassicauIis protoplasts could be obtained only when osmoticum was reduced gradually. Different combinations of plant growth regulators influenced the division of cultured protoplasts. 2,4-D (ling/l) in combination with 6BA(0.5mg/1) was the most suitable hormone conbination. If the level of 2,4-D and 6BA was higher than 2 mg/1, protoplast division was inhibited Plant regeneration via somatic embryogenesis. Protoplast-derived calti cultured on the differentiation medium (described in Materials and methods) turned embryogenic callus. Approximately three weeks later a yellowish and granular callus was formed on the surface of original calli (Fig. 1F). Careful selection of such embryogenic calli and their culture on hormonefree MS medium led to the formation of embryoids (Fig. 1G). Complete plantlets were obtained (Fig.H-I),
and grew in the greenhouse (Fig.lJ). Embryogenic calli have been subcultured on hormone-free MS medium for about one year, and they have maintain ed the ability to regenerate plantlets with nearly 100% differentiation frequency. But hundreds of regenerated plantlets, about 30% survived and grew normally.
Reference
I. Bhattacharya SK, Ghosal S, Chaudhuri RK, Simgh AK and Sharma PV (1974) J. Pharm.Sci. 63:13411342. 2. Davey MR, Bush E and Power JB (1974) Plant Sci. Letters 3:127-133. 3. Gosch G, Bajaj YPS and Reinert J (1975) Protoplasma 8 6 : 4 0 5 - 410. 4. Kao KN and Michayluk MR (1975) Planta 126:105110 5. Murashige T and Skoog F (1962) Physiol. Plant 15:473-493 6. Sadritdinov F (1971) In: Sultanov MB (eds) Farmakol.Alkaloidov serdechnykh. Glikozidov."Faff': Tashkent, USSR, pp146-148 7.Tulyaganov N, Danilevskii BL, Sadritdinov FS (1971) In:Sultanov MB (eds) Farmakol. Alkaloidov Serdech. Glikozidov."Fan":Tashkent, USSR.,pp148151 8. Zhou YL, Qian YQ, Cai QG, Zhang ZG and Yan X (1985) Acta Botanica Sinica 27(2): 148-150
