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Rapid Establishment of Tissue Cultures from Seeds of Panax ginseng and Panax pseudoginseng
Biochem. Physiol. Pflanzen 185, 131-134 (1989) VEB Gustav Fischer Verlag lena
Short Communication Rapid Establishment of Tissue Cultures from Seeds of Panax ginseng and Panax pseudoginseng ANN ODNEVALL l , LARS Bn)RK2 and TORKEL BERGLUND l 1
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden 1 Department of Pharmacognosy, BMC, Uppsala University, Uppsala, Sweden
Key Term Index: seed, tissues culture, gibberellic acid; Panax ginseng, Panax pseudoginseng
Summary Seeds from Panax ginseng S. A. MEYER and Panax pseudoginseng WALL. were used as starting material for tissue cultures. To achieve callus growth and shoot formation the seeds were surface sterilized and cut into 2 - 3 pieces. For Panax ginseng pretreatment with gibberellic acid was necessary to obtain growth. The pieces were placed on modified MS agar medium with different hormone combinations. Shoots and callus tissue were rapidly obtained. Fast growing suspension cultures were established from the calli.
Surface sterilized seeds are an excellent material for the establishment of tissue cultures. However in the Panax genus it is difficult to break the seed dormancy and different stratification programs including alternating cold and warm periods up to 18 months have been suggested. Gibberellic acid is well documented as an endogenously produced hormone which controls seed metabolism through formation and secretion of a great number of enzymes. Exogenous application of GA3 has been reported to stimulate the growth of Panax quinquefolium embryos. This paper demonstrates a method to break seed dormancy in P. ginseng and P. pseudoginseng. Plant Material
Seeds of Panax ginseng C. A. MEYER were obtained from Condon Enterprises, Wisconsin, USA. Seeds of Panax pseudoginseng WALL. was a gift from Botanischer Garten der Friedrich-SchillerUniversitat lena, GDR. Callus and Shoot Initiation
Seeds of P. ginseng were pelled mechanically while the seeds of P. pseudoginseng were used unpelled. The naked seed of P. ginseng was soaked in GAr solution for various periods according to Table 1. The seeds was then surface sterilized (5min in 70 % ethanol, 5-10 min in 5 % NaOCl), rinsed with sterile distilled water and cut into 2-3 pieces. The pieces were put on 30 ml solidified medium in 100 ml Erlenmeyer flasks and were kept under controlled conditions (25°C, light 12 hldark 12 h, 10 Wm- 2 Asea Skandia LRD 36 W/29. Abbreviations: BA, benzyladenine; 2,4-D, 2,4-dichlorophenoxyacetic acid; GA 3 , gibberellic acid; NAA, naphthaleneacetic acid; MS, Murashige-Skoog medium 9*
BPP 185 (1989) 112
131
Nutrient Media The medium used for callus and shoot initiation was a modified MS-medium according to GARVE et aI. (1980) supplemented with hormones according to Table 1 and 0.7% agar (Merck). The calli formed were further subcultivated on this medium supplemented with agar and 1 ppm 2,4-D and 1 NAA. The medium for suspension cultures was a mofidied MS-medium with 5mgl- 1 NAA and 0.02mgl- 1 kinetin.
Culture Conditions Callus culture were subcultured every 4 - 5 weeks. The suspension cultures were grown either in the light or in the dark in 100 ml medium in 250 ml Erlenmeyer flasks at 25°C, 135 rpm, stroke length 2.5 cm, subcultivation every 3-4 weeks.
Determination of Growth Parameters 2-4 g of fresh cells from a 2 weeks old suspension culture were transferred to fresh medium. The cells were harvesterd by filtration (fresh weight) and lyophilized (dry weight). One or two flasks were harvested every 3-4 days. The growth index (HI) is defined as the dry weight ofthe biomass divided by the dry weight of the inoculum. The specific growth rate ~ (day-I) is the calculated rate of the increase of biomass of a cell suspension per unit of biomass concentration during the exponential growth phase. (~= l/XidXldt where X = biomass concentration in g dry weightl- I).
Panax ginseng No germination was obtained with intact or intact peeled (the hard seedcoat removed) seeds. Pretreatment with GA3 had no effect in this case. However, if the seeds were cut into pieces after the GA3 treatment callus or shoot formation was obtained (cf. Table 1). the response to different concentrations of GA3 or different incubation times of the seeds varied strongly, possibly due to non identical pieces of the seeds used for inoculation. Medium I promoted callus growth and medium III shoot formation. This is consistent with the known effects of the growth factors in the media (2,4-D resp. BA). Medium II was not suitable for induction of neither callus nor shoots, possibly due to the absence of GA3 in this medium. However, when calli had grown to about 1 cm in diameter and were transferred to medium II they grew rapidly. Thus pretreatment of cut seeds with GA3 and inoculation on a GA3 containing substrate induce dormancy breakage and tissue culture formation of P. ginseng. Panax pseudoginseng The seeds of P. pseudoginseng formed callus rapidly after the cutting procedure. GA3 pretreatment was not necessary. Pieces inoculated on medium II formed small calli after about 1 month. These calli were subcultivated on medium II. Suspension Cultures Fast growing calli from both P. ginseng and P. pseudoginseng were transferred to a liquid medium. A suitable medium for rapid growth was modified MS supplemented with 5 ppm NAA and 0.02 ppm kinetin. The growth parameters for the cultures are shown in Table 2. The growth rate of P. ginseng was almost equal in light and darkness. f.t for P. pseudoginseng was higher in light than in darkness while the maximal GI values were almost equal. The lag phase was 2-3 days and the exponential phase shorter in light than in darkness for both species. 132
BPP 185 (1989) 112
Table l. Germination experiments with Panax ginseng seeds. Procedure described in Materials and Methods. 5 seedslflask in Exp . 1-3, repeated 4 times ; 5 seedslflask in Exp . 4- 20, repeated 2 tim es. (GA 3 ) ppm
Exp .
1 2 3 4 5 6 7 8 9 10
Days of incubation in GA3 solution
11 12 13 14 15 16 17 18 19 20
Growth after 3 months
germinating seeds
callus
++++ ++++
callus
++++
III I, II I, II, III I , III
shoots
+++
callus
+++ +
III I, III III III I
callus
++++
shoots shoots
+++ +++
I II III I , III
1-8
100 500 500 500 500 500 500 1000 1000 1000 1000 1000 1000 1500 1500 1500 1500
Medium*
1 2 2 5 8 1 1 2 5 8 8 1-5 6 8 8
II, III I II, III I, III I, III
* Medium I : MS + 1 ppm, 2,4-D + 0.04 ppm kinetin Medium II : MS + 1 ppm 2,4-D + 1 ppm NAA Medium III : 1/2 MS + 1 ppm GA3 + 1 ppm BA
+ 0.3 ppm GA3
Table 2 . Growth parameters f or suspension cutures of P. ginseng and P. p seudoginseng. Each value represent the mean from 3 separate experiments. Culture
Lightl dark
Maximal growth indo
f.t day-l
Lagphase days
Exp. phase days
P. ginseng
light dark
3.0 5.2
0.055 0 .063
3 3
17 23
P. pseudoginseng
light dark
9.3 8.2
0 .082 0 .067
2 3
18 25
Acknowledgement We want to thank the Nordic Industrial Foundation for financial support. BPP 185 (1989) 112
133
References GARVE, R., LUCKNER, M., VOGEL, E., TEWES, A., and NOVER, L.: Growth, morphogenesis and cardenolide formation in long-term cultures of Digitalis lanata. Planta Medica 40,92-103 (1980). MAYER, A. M., and SHAIN, Y.: Control of seed germination. Annu. Rev. Plant Physiol. 25, 167-193 (1974). SooN-KEUN HONG: Ginseng cultivation. In: Cultivation of Medical Plants (C. K. ATAL, B. M. KAPUR, eds.) pp. 418-435. United Printing Press, New Delhi 1982. THOMPSON, G. A., and MCCOWN, B.: Establishing a micropropagation system for American ginseng (Panax quinquejolium L.). Hort. Science 21,915 (1986).
Received December 5, 1988; accepted December 14, 1988 Authors' addresses: Dr. LARS BJORK (for correspondence), Department of Pharmacognosy, BMS, Uppsala University, S - 75123 Uppsala, Sweden; Dr. ANN ODNEVALL and Dr. TORKEL BERGLUND, Department of Biochemistry and Biotechnology, Royal Institute of Technology, S - 10044 Stockholm, Sweden.
134
BPP 185 (1989) 112
Short Communication Rapid Establishment of Tissue Cultures from Seeds of Panax ginseng and Panax pseudoginseng ANN ODNEVALL l , LARS Bn)RK2 and TORKEL BERGLUND l 1
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden 1 Department of Pharmacognosy, BMC, Uppsala University, Uppsala, Sweden
Key Term Index: seed, tissues culture, gibberellic acid; Panax ginseng, Panax pseudoginseng
Summary Seeds from Panax ginseng S. A. MEYER and Panax pseudoginseng WALL. were used as starting material for tissue cultures. To achieve callus growth and shoot formation the seeds were surface sterilized and cut into 2 - 3 pieces. For Panax ginseng pretreatment with gibberellic acid was necessary to obtain growth. The pieces were placed on modified MS agar medium with different hormone combinations. Shoots and callus tissue were rapidly obtained. Fast growing suspension cultures were established from the calli.
Surface sterilized seeds are an excellent material for the establishment of tissue cultures. However in the Panax genus it is difficult to break the seed dormancy and different stratification programs including alternating cold and warm periods up to 18 months have been suggested. Gibberellic acid is well documented as an endogenously produced hormone which controls seed metabolism through formation and secretion of a great number of enzymes. Exogenous application of GA3 has been reported to stimulate the growth of Panax quinquefolium embryos. This paper demonstrates a method to break seed dormancy in P. ginseng and P. pseudoginseng. Plant Material
Seeds of Panax ginseng C. A. MEYER were obtained from Condon Enterprises, Wisconsin, USA. Seeds of Panax pseudoginseng WALL. was a gift from Botanischer Garten der Friedrich-SchillerUniversitat lena, GDR. Callus and Shoot Initiation
Seeds of P. ginseng were pelled mechanically while the seeds of P. pseudoginseng were used unpelled. The naked seed of P. ginseng was soaked in GAr solution for various periods according to Table 1. The seeds was then surface sterilized (5min in 70 % ethanol, 5-10 min in 5 % NaOCl), rinsed with sterile distilled water and cut into 2-3 pieces. The pieces were put on 30 ml solidified medium in 100 ml Erlenmeyer flasks and were kept under controlled conditions (25°C, light 12 hldark 12 h, 10 Wm- 2 Asea Skandia LRD 36 W/29. Abbreviations: BA, benzyladenine; 2,4-D, 2,4-dichlorophenoxyacetic acid; GA 3 , gibberellic acid; NAA, naphthaleneacetic acid; MS, Murashige-Skoog medium 9*
BPP 185 (1989) 112
131
Nutrient Media The medium used for callus and shoot initiation was a modified MS-medium according to GARVE et aI. (1980) supplemented with hormones according to Table 1 and 0.7% agar (Merck). The calli formed were further subcultivated on this medium supplemented with agar and 1 ppm 2,4-D and 1 NAA. The medium for suspension cultures was a mofidied MS-medium with 5mgl- 1 NAA and 0.02mgl- 1 kinetin.
Culture Conditions Callus culture were subcultured every 4 - 5 weeks. The suspension cultures were grown either in the light or in the dark in 100 ml medium in 250 ml Erlenmeyer flasks at 25°C, 135 rpm, stroke length 2.5 cm, subcultivation every 3-4 weeks.
Determination of Growth Parameters 2-4 g of fresh cells from a 2 weeks old suspension culture were transferred to fresh medium. The cells were harvesterd by filtration (fresh weight) and lyophilized (dry weight). One or two flasks were harvested every 3-4 days. The growth index (HI) is defined as the dry weight ofthe biomass divided by the dry weight of the inoculum. The specific growth rate ~ (day-I) is the calculated rate of the increase of biomass of a cell suspension per unit of biomass concentration during the exponential growth phase. (~= l/XidXldt where X = biomass concentration in g dry weightl- I).
Panax ginseng No germination was obtained with intact or intact peeled (the hard seedcoat removed) seeds. Pretreatment with GA3 had no effect in this case. However, if the seeds were cut into pieces after the GA3 treatment callus or shoot formation was obtained (cf. Table 1). the response to different concentrations of GA3 or different incubation times of the seeds varied strongly, possibly due to non identical pieces of the seeds used for inoculation. Medium I promoted callus growth and medium III shoot formation. This is consistent with the known effects of the growth factors in the media (2,4-D resp. BA). Medium II was not suitable for induction of neither callus nor shoots, possibly due to the absence of GA3 in this medium. However, when calli had grown to about 1 cm in diameter and were transferred to medium II they grew rapidly. Thus pretreatment of cut seeds with GA3 and inoculation on a GA3 containing substrate induce dormancy breakage and tissue culture formation of P. ginseng. Panax pseudoginseng The seeds of P. pseudoginseng formed callus rapidly after the cutting procedure. GA3 pretreatment was not necessary. Pieces inoculated on medium II formed small calli after about 1 month. These calli were subcultivated on medium II. Suspension Cultures Fast growing calli from both P. ginseng and P. pseudoginseng were transferred to a liquid medium. A suitable medium for rapid growth was modified MS supplemented with 5 ppm NAA and 0.02 ppm kinetin. The growth parameters for the cultures are shown in Table 2. The growth rate of P. ginseng was almost equal in light and darkness. f.t for P. pseudoginseng was higher in light than in darkness while the maximal GI values were almost equal. The lag phase was 2-3 days and the exponential phase shorter in light than in darkness for both species. 132
BPP 185 (1989) 112
Table l. Germination experiments with Panax ginseng seeds. Procedure described in Materials and Methods. 5 seedslflask in Exp . 1-3, repeated 4 times ; 5 seedslflask in Exp . 4- 20, repeated 2 tim es. (GA 3 ) ppm
Exp .
1 2 3 4 5 6 7 8 9 10
Days of incubation in GA3 solution
11 12 13 14 15 16 17 18 19 20
Growth after 3 months
germinating seeds
callus
++++ ++++
callus
++++
III I, II I, II, III I , III
shoots
+++
callus
+++ +
III I, III III III I
callus
++++
shoots shoots
+++ +++
I II III I , III
1-8
100 500 500 500 500 500 500 1000 1000 1000 1000 1000 1000 1500 1500 1500 1500
Medium*
1 2 2 5 8 1 1 2 5 8 8 1-5 6 8 8
II, III I II, III I, III I, III
* Medium I : MS + 1 ppm, 2,4-D + 0.04 ppm kinetin Medium II : MS + 1 ppm 2,4-D + 1 ppm NAA Medium III : 1/2 MS + 1 ppm GA3 + 1 ppm BA
+ 0.3 ppm GA3
Table 2 . Growth parameters f or suspension cutures of P. ginseng and P. p seudoginseng. Each value represent the mean from 3 separate experiments. Culture
Lightl dark
Maximal growth indo
f.t day-l
Lagphase days
Exp. phase days
P. ginseng
light dark
3.0 5.2
0.055 0 .063
3 3
17 23
P. pseudoginseng
light dark
9.3 8.2
0 .082 0 .067
2 3
18 25
Acknowledgement We want to thank the Nordic Industrial Foundation for financial support. BPP 185 (1989) 112
133
References GARVE, R., LUCKNER, M., VOGEL, E., TEWES, A., and NOVER, L.: Growth, morphogenesis and cardenolide formation in long-term cultures of Digitalis lanata. Planta Medica 40,92-103 (1980). MAYER, A. M., and SHAIN, Y.: Control of seed germination. Annu. Rev. Plant Physiol. 25, 167-193 (1974). SooN-KEUN HONG: Ginseng cultivation. In: Cultivation of Medical Plants (C. K. ATAL, B. M. KAPUR, eds.) pp. 418-435. United Printing Press, New Delhi 1982. THOMPSON, G. A., and MCCOWN, B.: Establishing a micropropagation system for American ginseng (Panax quinquejolium L.). Hort. Science 21,915 (1986).
Received December 5, 1988; accepted December 14, 1988 Authors' addresses: Dr. LARS BJORK (for correspondence), Department of Pharmacognosy, BMS, Uppsala University, S - 75123 Uppsala, Sweden; Dr. ANN ODNEVALL and Dr. TORKEL BERGLUND, Department of Biochemistry and Biotechnology, Royal Institute of Technology, S - 10044 Stockholm, Sweden.
134
BPP 185 (1989) 112