- Email: [email protected]
Plant regeneration of Korean wild ginseng (Panax ginseng Meyer) mutant lines induced by γ-irradiation (60Co) of adventitious roots
Accepted Manuscript Plant regeneration of Korean wild ginseng (Panax ginseng Meyer) mutant lines 60 induced by γ-irradiation ( Co) of adventitious roots Jun-Ying Zhang, Hyeon-Jin Sun, In-Ja Song, Tae-Woong Bae, Hong-Gyu Kang, SukMin Ko, Yong-Ik Kwon, Il-Woung Kim, Jaechun Lee, Shin-Young Park, Pyung-Ok Lim, Yong Hwan Kim, Hyo-Yeon Lee PII:
S1226-8453(14)00051-7
DOI:
10.1016/j.jgr.2014.04.001
Reference:
JGR 29
To appear in:
Journal of Ginseng Research
Received Date: 13 December 2013 Revised Date:
24 March 2014
Accepted Date: 9 April 2014
Please cite this article as: Zhang J-Y, Sun H-J, Song I-J, Bae T-W, Kang H-G, Ko S-M, Kwon Y-I, Kim I-W, Lee J, Park S-Y, Lim P-O, Kim YH, Lee H-Y, Plant regeneration of Korean wild ginseng (Panax 60 ginseng Meyer) mutant lines induced by γ-irradiation ( Co) of adventitious roots, Journal of Ginseng Research (2014), doi: 10.1016/j.jgr.2014.04.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Title: Plant regeneration of Korean wild ginseng (Panax ginseng Meyer)
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mutant lines induced by γ-irradiation (60Co) of adventitious roots
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Running title: Regeneration of mutant adventitious roots
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Jun-Ying Zhang1†, Hyeon-Jin Sun2†, In-Ja Song2, Tae-Woong Bae2, Hong-Gyu Kang2, Suk-
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Min Ko2, Yong-Ik Kwon2, Il-Woung Kim2, Jaechun Lee3, Shin-Young Park4, Pyung-Ok Lim5,
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Yong Hwan Kim6**, and Hyo-Yeon Lee1,2*
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Faculty of Biotechnology, Jeju National University, Jeju 690-756, Korea
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Subtropical Horticulture Research Institute, Jeju National University, Jeju 690-756, Korea
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School of Medicine, Jeju National University, Jeju 690-756, Korea
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Department of Clinical Pathology, Cheju Halla University, Jeju 690-708, Korea
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Department of New Biology, Daegu Gyeongbuk Institute of Science and Technology
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(DGIST), Daegu 711-873, Korea
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and Fisheries (IPET), Anyang 431-810, Korea
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Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry
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†
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These authors contributed equally to this work.
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*, **
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Corresponding authors
E-mail: [email protected] E-mail: [email protected]
Tel: +82-64-754-3347, Fax: +82-64-754-3890 Tel: +82-31-420-6731, Fax: +82-31-425-6441
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ABSTRACT We established an efficient in vitro protocol for somatic embryogenesis and plantlet
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conversion of Korean wild ginseng (Panax ginseng Meyer). Wild-type and mutant
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adventitious roots derived from the ginseng produced calli on Murashige and Skoog (MS)
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medium supplemented with 0.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.3 mg/L
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kinetin; 53.3% of the explants formed callus. Embryogenic callus proliferation and somatic
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embryo induction occurred on MS medium containing 0.5 mg/L 2,4-D. The induced somatic
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embryos further developed to maturity on MS medium with 5 mg/L gibberellic acid (GA3)
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and 85% of them germinated. The germinated embryos were developed to shoots and
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elongated on MS medium with 5 mg/L GA3. The shoots developed into plants with well-
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developed taproots on 1/3 strength Schenk and Hildebrandt (SH) basal medium supplemented
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with 0.25 mg/L 1-naphthaleneacetic acid (NAA). When the plants transferred to soil, about
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30% of the regenerated plants developed into normal plants.
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Keywords: Panax ginseng Meyer, Somatic embryogenesis, Somatic embryo, Mutant
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adventitious roots
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INTRODUCTION Panax ginseng Meyer is an important medicinal herb and is widely cultivated in Korea,
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China and Japan. The root has been used as a drug for over 2000 years in oriental countries.
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Its use is rapidly expanding in the Western countries as complementary and alternative
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medicine [1]. Ginsenosides are the major pharmacologically active components in P. ginseng.
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More than 30 types of ginsenosides have been identified from the genus [2-3].
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Ginseng is a perennial plant which grows slowly and has a long production cycle (4-6
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years). And more than 3 years of juvenile period are required for producing seeds [4-5]. This
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has made the generation of superior genotypes by conventional breeding very difficult.
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Because of these reasons, attempts have been made to achieve a more rapid and increased
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production of the ginsenosides using other methods such as classical tissue culture system [6],
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bioreactor culture system [7], Agrobacterium-mediated hairy root production [8-9], using
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elicitors in cell cultures [10-12], and mutation breeding by γ-irradiation [13-14]. The last
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method has been used in many other plant species and has provided a large number of
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variants useful for plant breeding [15-17]. Mutagenesis by γ-irradiation enhanced ginsenoside
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production in P. ginseng [13-14]. Recently, we have also generated mutant cell lines by
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applying γ-irradiation on P. ginseng adventitious roots which were derived from Korean wild
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ginseng root [18]. Among the selected mutant cell lines, line 1 has showed the highest total
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ginsenoside content of 7 major ginsenosides (Rg1, Re, Rb1, Rb2, Rc, Rf, and Rd). The total
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ginsenoside content of the mutant line was 2.3 times higher than in the wild-type line [18].
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Using γ-irradiation, we have created a useful mutant line for breeding of the ginseng plant.
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However, there are no reports on in vitro plant regeneration with mutant lines of ginseng
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adventitious root.
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Plant tissue culture system is considered a valuable tool in the plant improvement 3
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propagation of many plant species [19-21]. P. ginseng is a difficult species to manipulate in
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vitro; however, its regeneration has generally been accomplished using somatic
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embryogenesis in callus derived from mature root tissues [22-24], callus derived from zygotic
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embryo [25-26], protoplast derived from callus [27], and cotyledons [4,28-30]. The
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development of efficient in vitro culture methods has facilitated the use of mutation technique
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for improvement of vegetative propagation of ginseng adventitious roots [13-14,18]. At
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present no information is available on the regeneration of mutant adventitious root line that
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has been selected from γ-irradiated P. ginseng adventitious roots.
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In this paper, we report on an efficient procedure for the regeneration of wild-type and mutant cell lines of P. ginseng adventitious roots through somatic embryogenesis.
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MATERIALS AND METHODS
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Callus induction and proliferation
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Adventitious roots derived from Korean wild ginseng were provided by Sunchon
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National University. The adventitious roots were generated as described previously [7, 31-32]
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and have been maintained in our laboratory for over 10 years. A mutant adventitious root line
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has been generated from the wild type adventitious roots by γ-irradiation [18]. For
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embryogenic callus induction, wild-type and mutant adventitious roots were sectioned into 10
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mm in length and were placed on MS solid medium supplemented with 2, 4-D, kinetin and 3%
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sucrose. The media were solidified with 0.3% Gelite. Callus induction frequency was tested
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on MS solid medium supplemented with various concentrations of 2, 4-D (0.5, 1, 1.5, 2 mg/L)
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and kinetin (0, 0.3, 0.5 mg/L). All media were adjusted to pH 5.8 before autoclaving. Thirty
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pieces of adventitious roots were placed on each petri dish. Three replicates were prepared for
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each treatment. All cultures were incubated at 25
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observed after 4 weeks of culture. After 6 weeks of culture, the frequency of callus induction
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was estimated. The induced callus was subcultured at 3 week intervals on the same medium for induction of embryogenic callus and maintenance.
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Induction of somatic embryos
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in the dark. Callus formation was
Embryogenic callus induced from the segments of adventitious roots was used for
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induction of somatic embryos. Ten g of embryogenic callus was incubated in a 15 L airlift
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bioreactor containing 5 L MS liquid medium with 0.5 mg/L 2, 4-D and 3% sucrose for
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proliferation. After 3 weeks, the proliferated embryogenic callus was used as explants for
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induction of somatic embryogenesis.
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To examine the effect of 2,4-D on somatic embryo induction, proliferated callus was
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placed on a solid MS medium supplemented with different concentrations of 2,4-D (0, 0.5, 1
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mg/L). Ten clumps of embryogenic callus (about 5mm in diameter) were cultured on petri
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dishes containing 40 mL of medium and the experiment repeated three times. All cultures
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were incubated at 25
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examined after 6 weeks of culture by counting cultured embryogenic callus that formed
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somatic embryos.
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in the dark. The frequency of somatic embryo production was
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When the callus produced globular stage embryos on MS solid medium with 2,4-D and 3%
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sucrose, the globular embryos were removed and transferred to 500 mL-Erlenmeyer flasks
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containing 200 mL of liquid MS medium supplemented with 2,4-D and 3% sucrose for
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further growth. The liquid cultures were agitated at 100 rpm on a gyratory shaker in the dark.
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After 1 month of culture, the proliferated globular embryos in flasks were transferred to each 5
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petri dish containing solid MS medium with GA3 and 3% sucrose for maturation and
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germination of embryos.
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Maturation and germination of somatic embryos The proliferated globular embryos in flasks were transferred to 40 mL MS solid medium
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supplemented with GA3 and 3% sucrose in 100 x 20 mm plastic petri dishes for maturation
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and germination. To investigate the effect of GA3 concentration on maturation and
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germination of somatic embryos, 150 globular embryos were transferred to germination
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medium containing 0, 1, 3, 5, 7, or 10 mg/L of GA3. Cultures were maintained at 23 ± 2
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under dim light illumination (12 µmol m-2 s-1) with a 16/8 h (light/dark) photoperiod. After 6
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weeks of culture, maturation and germination of embryos were examined. The experiment
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was repeated three times.
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Development of plantlets and acclimatization When shoots reached 0.5-1.0 cm in height, the plantlets were transferred from
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germination medium to elongation medium, 50 mL MS solid medium supplemented with 5
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mg/L GA3 in 100×40 mm plastic petri dishes, for shoot elongation. When shoots grew 3.0-
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4.0 cm in height, they were transferred to rooting medium, half or one-third strength MS, or
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SH basal medium supplemented with 0.25 mg/L NAA or with 0.5% activated charcoal, in
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75×130 mm glass bottles, one shoot per bottle. Cultures were conducted in a culture room
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and maintained in a 16/8 h (light/dark) photoperiod with white fluorescent light (30 µmol m-2
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s-1) at 23 ± 2 . After 4 weeks, the results of rooting were examined.
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Plantlets with both shoots and roots were transferred to plastic pots (10×18 cm)
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containing an artificial soil mixture of peat moss, vermiculite and perlite (2:3:1 v/v) and 6
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(40 µmol m-2s-1, 16 h photoperiod, 25 ± 1 ). After 3 weeks, the plants were hardened by
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removing the polyvinyl film gradually on a daily basis for one week, and then the film was
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removed. After 3 months of culture, the survived plants without wilting were counted. The
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acclimated plants were transplanted to glasshouse conditions or kept in the growth room for
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another 4-6 months.
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Data analysis
Each of the treatments was performed three times. Statistical analyses were performed
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according to the one-way analysis of variance (ANOVA) using SPSS software (version 17.0)
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to assess significant differences in the mean values of different treatments. Comparisons
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between the mean values were assessed using Duncan’s multiple-range test (P < 0.05).
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RESULTS AND DISCUSSION
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Induction of callus from adventitious roots
Initiation of callus from adventitious root explants generally occurred after 3 weeks on
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media supplemented with different combinations of growth regulators. The highest frequency
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of callus induction was observed on the medium containing 0.5 mg/L 2,4-D and 0.3 mg/L
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kinetin. The frequency of callus induction reduced dramatically as the concentration of 2,4-D
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increased. Callus was not induced in the presence of 2 mg/L 2,4-D (Table 1). Similar results
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were reported with the cultures of hairy roots of P. ginseng that 2,4-D at more than 3 mg/L
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strongly suppressed callus induction [33]. When the segments of adventitious roots (Fig. 1A)
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of P. ginseng were incubated in MS solid medium with 0.5 mg/L 2, 4-D and 0.3 mg/L kinetin,
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callus was induced from the cut sides of the adventitious roots after 6 weeks of culture (Fig.
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three months, embryogenic callus was induced (Fig. 1C) and the embryogenic callus showed
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high regenerative capacity and differentiated into somatic embryos and plantlets. Callus
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induction and growth from adventitious root explants was dependent upon 2,4-D as
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previously reported [22,24,27]. When embryogenic callus was transferred to MS medium
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lacking kinetin, a small number of globular embryos formed after 3 weeks of culture (Fig. 1D,
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E). Thus, it is essential to induce and maintain the embryogenic callus in the medium
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supplemented with 2,4-D in combination with kinetin. Embryogenic callus has been
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maintained in the dark for more than 2 years through 3-week subculture intervals on MS solid
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medium with 0.5 mg/L 2,4-D and 0.3 mg/L kinetin.
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Induction of somatic embryos
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The embryogenic callus grows better in a liquid medium than a solid medium (data not
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shown). Therefore, we propagated the embryogenic callus in bioreactor to assess somatic
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embryo development and plantlet conversion. When embryogenic callus was inoculated into
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a 15 L airlift bioreactor containing 5 L MS liquid medium with 0.5 mg/L 2,4-D, the
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embryogenic callus was propagated and a small number of globular shaped embryos were
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also formed after 3 weeks of culture (Fig. 1D, E). The growth rate (final explant fresh
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weight/initial explant fresh weight) was about 2.1. Embryogenic cell clumps proliferated in
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bioreactor were transferred onto MS solid medium with different concentrations of 2,4-D (0,
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0.5 or 1.0 mg/L) for embryogenesis. The frequency of somatic embryo formation was
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significantly depended on the concentrations of 2,4-D (Table 2). The highest induction
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frequency of somatic embryos was observed on the medium supplemented with 0.5 mg/L
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2,4-D. The frequency of somatic embryo formation in wild-type and mutant cell line was
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in wild-type and mutant cell line, respectively. There was no significant difference in somatic
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embryo formation frequency between wild-type and mutant cell line (Table 2). Globular
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shaped somatic embryos formed on the surfaces of embryogenic callus (Fig. 1F, G).
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Maturation and germination of somatic embryos
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These somatic embryos were transferred into 500 mL-Erlenmeyer flasks containing 200
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mL of liquid MS medium supplemented with 0.5 mg/L 2,4-D and 3% sucrose (Fig. 1H) for
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proliferation. The growth rate (final explant fresh weight/initial explant fresh weight) was
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about 1.7. After 4 weeks of culture, the proliferated globular embryos were transferred to
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petri dishes containing solid MS medium with various concentrations of GA3 and 3% sucrose.
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At 5 mg/L GA3, most of the globular embryos turned green and increased in size and
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developed into torpedo and cotyledonary stage embryos within one month. When the mature
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somatic embryos were transferred to a fresh medium with the same composition, most of the
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embryos germinated within 2 weeks of culture (Fig. 1I). Adventitious shoots were induced
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from the mature somatic embryos. The optimal concentration of GA3 in germination medium
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was 5 mg/L, yielding the highest germination frequency of 85%. Without GA3 treatment, the
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germination frequency was lowest at 36%. Maturation and germination of embryos were
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strongly influenced by the GA3 concentration (Table 3). This result suggests that GA3 is
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required for maturation and germination of somatic embryos. Similar results were observed
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in Eleutherococcus senticosus, that GA3 treatment was necessary to induce germination from
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somatic embryos [34]. GA3 treatment is also commonly used for maturation and germination
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of somatic embryos from P. ginseng [22,26,28-29], from P. quinquefolius [35] and from P.
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japonicus [36].
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Development of plantlets and transplantation When shoots reached 0.5-1.0 cm in height on germination medium, the shoots were
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transferred to elongation medium, 50 mL MS solid medium supplemented with 5 mg/L GA3
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in 100×40 mm plastic petri dishes, for further growth of shoots. After about one month of
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culture, the shoots developed to 3.0-4.0 cm in height, but most of the shoots had no visible
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roots. The shoots without roots were excised and transferred to different rooting media, half
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or one-third strength MS, or SH basal medium supplemented with 0.25 mg/L NAA or with
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0.5% activated charcoal, in 75×130 mm glass bottles, one shoot per bottle. Adventitious roots
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formed from the excised regions of the shoots. After 1 month, the rate of root formation from
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the shoots was examined (Table 4). As far as root quality is concerned, 1/3 SH medium with
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0.25 mg/L NAA and 1% sucrose showed the best result among the tested rooting media; the
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roots grew fast and thickened on the medium (Fig. 1J; Fig. 2A, B; Table 4). Although 1/3 SH
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medium with 2% sucrose and 0.5% activated charcoal was most effective in inducing roots,
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the roots grew well but weak (Table 4). The optimal medium for rooting is therefore 1/3 SH
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medium supplemented with 0.25 mg/L NAA and 1% sucrose among the tested rooting media
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in this study. In our comparative studies, SH medium was more effective than MS medium in
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root induction and proliferation. A very similar result was reported in American [35] and
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Korean ginsengs [30]. It was reported that the high level of ammonium nitrate in MS medium
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highly suppressed root development in carrot [37]. Choi et al. [5] reported that when the
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ammonium nitrate was omitted in MS medium, root growth of regenerated ginseng plants
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was enhanced. The concentration of ammonium nitrate in SH medium was about 8 times
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lower than in MS medium. It seems that the different concentrations of ammonium nitrate in
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SH and MS medium may result in the different root induction efficiency between the two
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basal medium. From these observations, we suggest that SH medium, especially 1/3 strength
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SH medium is suitable for root induction and growth of regenerated ginseng plants. Well-developed plantlets with both shoots and roots derived from adventitious roots were
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transferred to plastic pots (10×18 cm) containing an artificial soil mixture of peat moss,
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vermiculite and perlite (2:3:1 v/v) in a growth room (Fig. 2C). The survival rate of the
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plantlets was about 30% after 3 months of culture and new leaf began growing (Fig. 2D). The
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plants regenerated from both wild-type and mutant cell line acclimatized in the growth room
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(Fig. 3).
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In conclusion, we developed an efficient in vitro regeneration protocol for an important
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medicinal plant of P. ginseng. The protocol described here will allow a relatively rapid mass
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production of Korean wild ginseng plants. It takes 6-8 months from the callus induction of
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adventitious roots to the plantation of plants. In the present study, we also produced the
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regenerated plants from the mutant adventitious roots which were obtained by γ-irradiation.
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The combination of mutation technique by γ-irradiation and plant regeneration by tissue
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cultures may be an effective way to ginseng improvement. The protocol established in this
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study is currently being used for the genetic transformation of this species.
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ACKNOWLEDGEMENTS This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2009-0094059).
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26. Arya S, Arya ID, Eriksson T. Rapid multiplication of adventitious somatic embryos of Panax ginseng. Plant Cell Tiss Organ Cult 1993;34:157-162. 27. Arya S, Liu JR, Eriksson T. Plant regeneration from protoplasts of Panax ginseng (C.A.
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Meyer) through somatic embryogenesis. Plant Cell Rep 1991;10:277-281.
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28. Choi YE, Yang DC, Yoon ES, Choi KT. Plant regeneration via adventitious bud
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formation from cotyledon explants of Panax ginseng C. A. Meyer. Plant Cell Rep
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1998;17:731-736.
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29. Choi YE, Yang DC, Yoon ES, Choi KT. High-efficiency plant production via direct
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somatic single embryogenesis from preplasmolysed cotyledons of Panax ginseng and 14
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possible dormancy of somatic embryos. Plant Cell Rep 1999;18:493-499. 30. Kim YJ, Lee OR, Kim KT, Yang DC. High frequency of plant regeneration through
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cyclic secondary somatic embryogenensis in Panax ginseng. J Ginseng Res 2012;36:442-
339
448.
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31. Yu KW, Gao W, Hahn EJ, Paek KY. Jasmonic acid improves ginsenoside accumulation
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in adventitious root culture of Panax ginseng C.A. Meyer. Biochem Eng J 2002;11:211-
342
215.
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32. Kim YS, Hahn EJ, Yeung EC, Paek KY. Lateral root development and saponin
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accumulation as affected by IBA or NAA in adventitious root cultures of Panax ginseng
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C.A. Meyer. In Vitro Cell Dev Biol Plant 2003;39:245-249.
346 347
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33. Kwon JH, Cheon HC, Yang DC. Production of ginsenoside in callus of ginseng hairy roots. J Ginseng Res 2003;27:78-85.
34. Choi YE, Kim JW, Yoon ES. High frequency of plant production via somatic
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embryogenesis from callus of cell suspension cultures in Eleutherococcus senticocus.
350
Ann Bot 1999;83:309-314.
353 354 355 356
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35. Zhou S, Brown DCW. High efficiency plant production of North American ginseng via somatic embryogenesis from cotyledon explants. Plant Cell Rep 2006;25:166-173. 36. You XL, Han JY, Choi YE. Plant regeneration via direct somatic embryogenesis in Panax
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japonicas. Plant Biotechnol Rep 2007;1:5-9. 37. Halperin W. Alternative morphogenetic events in cell suspensions. Am J Bot 1966;53:443-453.
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TABLES AND FIGURES
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Table 1. Effects of 2,4-D and kinetin on the frequency of callus formation from ginseng
363
adventitious roots
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2, 4-D
Kinetin
Number of
Number of root explants
(mg/L)
(mg/L)
root explants
formed callus
0.5
0
30
4.3 ±1.0de
0.3
30
0.5
30
0
30
0.3
30
0.5 2
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9.1 ± 1.7de 7.2 ± 1.2bc
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16.2 ± 1.8a
0 0.3 0.5
30
2.3 ± 0.8ef
30
0.0 ± 0f
30
0.0 ± 0f
30
0.0 ± 0f
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5.4 ± 1.1cd
The data were collected after 6 weeks of culture.
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The results represent the means ± SEM of values obtained from three experiments.
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Different corresponding letters within a column are significant different at P<0.05 by
368
Duncan’s multiple range test.
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Figure 1. Somatic embryogenesis and regeneration of plantlet from adventitious roots of
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Panax ginseng. (A) Adventitious roots derived from Korean wild ginseng root. (B) Callus
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induction from adventitious root explants. (C) Embryogenic callus derived from adventitious
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roots. (D) Proliferation of embryogenic callus in an airlift bioreactor. (E) Proliferated
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embryogenic cell clumps from bioreactor culture. (F) Somatic embryos formed on
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embryogenic callus. (G) Magnified image from (F) (scale bar = 2 mm). (H) Proliferation of
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somatic embryos in conical flasks. (I) Maturation and germination of somatic embryos on MS
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medium supplemented with 5 mg/L GA3. (J) Well-developed plantlet derived from somatic
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embryo (scale bar = 0.8 cm).
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Table 2. Effects of 2,4-D on somatic embryo formation from embryogenic callus of wild-type
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and mutant adventitious roots
Mutant
Frequency of somatic
Number of somatic
(mg/L)
embryo formation (%)
embryos per callus
0
5.3 ± 0.73b
10.0 ± 1.2b
0.5
15.3 ± 1.21a
1
2.5 ± 0.46c
0
5.8 ± 0.28b
0.5
14.7 ± 0.45a
1
2.2 ± 0.27c
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Wild-type
2,4-D
25.6 ± 2.3a 4.7 ± 0.3c
12.3 ± 1.9b 23.7 ± 0.6a
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Cell line
6.0 ± 1.4c
The data were collected after 6 weeks of culture.
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The results represent the means ± SEM of values obtained from three experiments.
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Different corresponding letters within a column are significant different at P<0.05 by
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Duncan’s multiple range test.
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Number of somatic
Germination frequency
(mg/L)
embryos inoculated
embryos germinated
(%)
0
150
53 ± 6c
36 ± 4c
1
150
60 ± 9c
40 ± 6c
3
150
66 ± 10bc
5
150
127 ± 7a
7
150
75 ± 6b
10
150
65 ± 5bc
45 ± 7bc 85 ± 5a
50 ± 4b
44 ± 3bc
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Concentration of GA3
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The data were collected after 6 weeks of culture on MS medium with 3% sucrose.
415
The results represent the means ± SEM of values obtained from three experiments.
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Different corresponding letters within a column are significant different at P<0.05 by
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Duncan’s multiple range test.
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Root frequency
No. of roots
Description on
shoots
(%)
per plant
root quality
1/2 MS 3% Sucrose
30
36
1.6
Grows slow, calluses
1/2 MS + 3% Sucrose + 0.5% charcoal
30
45
1.0
Grows slow
1/3 MS + 1% Sucrose + 0.25 mg/L NAA
30
58
1/2 SH + 3% Sucrose
30
71
1/2 SH + 2% Sucrose + 0.5% charcoal
30
62
1/3 SH + 2% Sucrose + 0.5% charcoal
30
80
1/3 SH + 1% Sucrose + 0.25 mg/L NAA
30
76
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445
447 448 449
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452
Grows fast, thin
1.0
Grows slow
1.0
Grows slow, calluses
1.0
Grows fast, thin
1.2
Grows fast, strong
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1.0
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Media
459 460 461 21
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Figure 2. Development of ginseng plant and transplantation into soil. (A) Well-developed
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plantlet on rooting medium, 1/3 SH basal medium supplemented with 0.25 mg/L NAA. (B)
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Plant with a taproot just before transplantation into soil. (C) Regenerated plant hardened in
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soil. (D) New leaves (indicated by arrows) were produced from three-month old potted plant
432
(scale bar = 0.8 cm).
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Figure 3. Transplantation of regenerated ginseng plants into soil. (A-E) Plants derived from
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wild-type adventitious roots. (F-J) Plants derived from mutant adventitious roots.
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S1226-8453(14)00051-7
DOI:
10.1016/j.jgr.2014.04.001
Reference:
JGR 29
To appear in:
Journal of Ginseng Research
Received Date: 13 December 2013 Revised Date:
24 March 2014
Accepted Date: 9 April 2014
Please cite this article as: Zhang J-Y, Sun H-J, Song I-J, Bae T-W, Kang H-G, Ko S-M, Kwon Y-I, Kim I-W, Lee J, Park S-Y, Lim P-O, Kim YH, Lee H-Y, Plant regeneration of Korean wild ginseng (Panax 60 ginseng Meyer) mutant lines induced by γ-irradiation ( Co) of adventitious roots, Journal of Ginseng Research (2014), doi: 10.1016/j.jgr.2014.04.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Title: Plant regeneration of Korean wild ginseng (Panax ginseng Meyer)
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mutant lines induced by γ-irradiation (60Co) of adventitious roots
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Running title: Regeneration of mutant adventitious roots
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Jun-Ying Zhang1†, Hyeon-Jin Sun2†, In-Ja Song2, Tae-Woong Bae2, Hong-Gyu Kang2, Suk-
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Min Ko2, Yong-Ik Kwon2, Il-Woung Kim2, Jaechun Lee3, Shin-Young Park4, Pyung-Ok Lim5,
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Yong Hwan Kim6**, and Hyo-Yeon Lee1,2*
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1
Faculty of Biotechnology, Jeju National University, Jeju 690-756, Korea
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2
Subtropical Horticulture Research Institute, Jeju National University, Jeju 690-756, Korea
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3
School of Medicine, Jeju National University, Jeju 690-756, Korea
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4
Department of Clinical Pathology, Cheju Halla University, Jeju 690-708, Korea
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5
Department of New Biology, Daegu Gyeongbuk Institute of Science and Technology
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(DGIST), Daegu 711-873, Korea
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6
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and Fisheries (IPET), Anyang 431-810, Korea
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Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry
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†
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These authors contributed equally to this work.
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*, **
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*
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**
Corresponding authors
E-mail: [email protected] E-mail: [email protected]
Tel: +82-64-754-3347, Fax: +82-64-754-3890 Tel: +82-31-420-6731, Fax: +82-31-425-6441
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ABSTRACT We established an efficient in vitro protocol for somatic embryogenesis and plantlet
27
conversion of Korean wild ginseng (Panax ginseng Meyer). Wild-type and mutant
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adventitious roots derived from the ginseng produced calli on Murashige and Skoog (MS)
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medium supplemented with 0.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.3 mg/L
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kinetin; 53.3% of the explants formed callus. Embryogenic callus proliferation and somatic
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embryo induction occurred on MS medium containing 0.5 mg/L 2,4-D. The induced somatic
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embryos further developed to maturity on MS medium with 5 mg/L gibberellic acid (GA3)
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and 85% of them germinated. The germinated embryos were developed to shoots and
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elongated on MS medium with 5 mg/L GA3. The shoots developed into plants with well-
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developed taproots on 1/3 strength Schenk and Hildebrandt (SH) basal medium supplemented
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with 0.25 mg/L 1-naphthaleneacetic acid (NAA). When the plants transferred to soil, about
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30% of the regenerated plants developed into normal plants.
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Keywords: Panax ginseng Meyer, Somatic embryogenesis, Somatic embryo, Mutant
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adventitious roots
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INTRODUCTION Panax ginseng Meyer is an important medicinal herb and is widely cultivated in Korea,
51
China and Japan. The root has been used as a drug for over 2000 years in oriental countries.
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Its use is rapidly expanding in the Western countries as complementary and alternative
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medicine [1]. Ginsenosides are the major pharmacologically active components in P. ginseng.
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More than 30 types of ginsenosides have been identified from the genus [2-3].
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Ginseng is a perennial plant which grows slowly and has a long production cycle (4-6
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years). And more than 3 years of juvenile period are required for producing seeds [4-5]. This
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has made the generation of superior genotypes by conventional breeding very difficult.
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Because of these reasons, attempts have been made to achieve a more rapid and increased
59
production of the ginsenosides using other methods such as classical tissue culture system [6],
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bioreactor culture system [7], Agrobacterium-mediated hairy root production [8-9], using
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elicitors in cell cultures [10-12], and mutation breeding by γ-irradiation [13-14]. The last
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method has been used in many other plant species and has provided a large number of
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variants useful for plant breeding [15-17]. Mutagenesis by γ-irradiation enhanced ginsenoside
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production in P. ginseng [13-14]. Recently, we have also generated mutant cell lines by
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applying γ-irradiation on P. ginseng adventitious roots which were derived from Korean wild
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ginseng root [18]. Among the selected mutant cell lines, line 1 has showed the highest total
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ginsenoside content of 7 major ginsenosides (Rg1, Re, Rb1, Rb2, Rc, Rf, and Rd). The total
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ginsenoside content of the mutant line was 2.3 times higher than in the wild-type line [18].
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Using γ-irradiation, we have created a useful mutant line for breeding of the ginseng plant.
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However, there are no reports on in vitro plant regeneration with mutant lines of ginseng
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adventitious root.
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Plant tissue culture system is considered a valuable tool in the plant improvement 3
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propagation of many plant species [19-21]. P. ginseng is a difficult species to manipulate in
75
vitro; however, its regeneration has generally been accomplished using somatic
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embryogenesis in callus derived from mature root tissues [22-24], callus derived from zygotic
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embryo [25-26], protoplast derived from callus [27], and cotyledons [4,28-30]. The
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development of efficient in vitro culture methods has facilitated the use of mutation technique
79
for improvement of vegetative propagation of ginseng adventitious roots [13-14,18]. At
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present no information is available on the regeneration of mutant adventitious root line that
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has been selected from γ-irradiated P. ginseng adventitious roots.
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In this paper, we report on an efficient procedure for the regeneration of wild-type and mutant cell lines of P. ginseng adventitious roots through somatic embryogenesis.
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MATERIALS AND METHODS
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Callus induction and proliferation
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Adventitious roots derived from Korean wild ginseng were provided by Sunchon
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National University. The adventitious roots were generated as described previously [7, 31-32]
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and have been maintained in our laboratory for over 10 years. A mutant adventitious root line
90
has been generated from the wild type adventitious roots by γ-irradiation [18]. For
91
embryogenic callus induction, wild-type and mutant adventitious roots were sectioned into 10
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mm in length and were placed on MS solid medium supplemented with 2, 4-D, kinetin and 3%
93
sucrose. The media were solidified with 0.3% Gelite. Callus induction frequency was tested
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on MS solid medium supplemented with various concentrations of 2, 4-D (0.5, 1, 1.5, 2 mg/L)
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and kinetin (0, 0.3, 0.5 mg/L). All media were adjusted to pH 5.8 before autoclaving. Thirty
96
pieces of adventitious roots were placed on each petri dish. Three replicates were prepared for
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each treatment. All cultures were incubated at 25
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observed after 4 weeks of culture. After 6 weeks of culture, the frequency of callus induction
99
was estimated. The induced callus was subcultured at 3 week intervals on the same medium for induction of embryogenic callus and maintenance.
101 102
Induction of somatic embryos
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in the dark. Callus formation was
Embryogenic callus induced from the segments of adventitious roots was used for
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induction of somatic embryos. Ten g of embryogenic callus was incubated in a 15 L airlift
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bioreactor containing 5 L MS liquid medium with 0.5 mg/L 2, 4-D and 3% sucrose for
106
proliferation. After 3 weeks, the proliferated embryogenic callus was used as explants for
107
induction of somatic embryogenesis.
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To examine the effect of 2,4-D on somatic embryo induction, proliferated callus was
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placed on a solid MS medium supplemented with different concentrations of 2,4-D (0, 0.5, 1
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mg/L). Ten clumps of embryogenic callus (about 5mm in diameter) were cultured on petri
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dishes containing 40 mL of medium and the experiment repeated three times. All cultures
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were incubated at 25
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examined after 6 weeks of culture by counting cultured embryogenic callus that formed
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somatic embryos.
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in the dark. The frequency of somatic embryo production was
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When the callus produced globular stage embryos on MS solid medium with 2,4-D and 3%
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sucrose, the globular embryos were removed and transferred to 500 mL-Erlenmeyer flasks
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containing 200 mL of liquid MS medium supplemented with 2,4-D and 3% sucrose for
118
further growth. The liquid cultures were agitated at 100 rpm on a gyratory shaker in the dark.
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After 1 month of culture, the proliferated globular embryos in flasks were transferred to each 5
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petri dish containing solid MS medium with GA3 and 3% sucrose for maturation and
121
germination of embryos.
122 123
Maturation and germination of somatic embryos The proliferated globular embryos in flasks were transferred to 40 mL MS solid medium
125
supplemented with GA3 and 3% sucrose in 100 x 20 mm plastic petri dishes for maturation
126
and germination. To investigate the effect of GA3 concentration on maturation and
127
germination of somatic embryos, 150 globular embryos were transferred to germination
128
medium containing 0, 1, 3, 5, 7, or 10 mg/L of GA3. Cultures were maintained at 23 ± 2
129
under dim light illumination (12 µmol m-2 s-1) with a 16/8 h (light/dark) photoperiod. After 6
130
weeks of culture, maturation and germination of embryos were examined. The experiment
131
was repeated three times.
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Development of plantlets and acclimatization When shoots reached 0.5-1.0 cm in height, the plantlets were transferred from
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germination medium to elongation medium, 50 mL MS solid medium supplemented with 5
136
mg/L GA3 in 100×40 mm plastic petri dishes, for shoot elongation. When shoots grew 3.0-
137
4.0 cm in height, they were transferred to rooting medium, half or one-third strength MS, or
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SH basal medium supplemented with 0.25 mg/L NAA or with 0.5% activated charcoal, in
139
75×130 mm glass bottles, one shoot per bottle. Cultures were conducted in a culture room
140
and maintained in a 16/8 h (light/dark) photoperiod with white fluorescent light (30 µmol m-2
141
s-1) at 23 ± 2 . After 4 weeks, the results of rooting were examined.
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Plantlets with both shoots and roots were transferred to plastic pots (10×18 cm)
143
containing an artificial soil mixture of peat moss, vermiculite and perlite (2:3:1 v/v) and 6
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145
(40 µmol m-2s-1, 16 h photoperiod, 25 ± 1 ). After 3 weeks, the plants were hardened by
146
removing the polyvinyl film gradually on a daily basis for one week, and then the film was
147
removed. After 3 months of culture, the survived plants without wilting were counted. The
148
acclimated plants were transplanted to glasshouse conditions or kept in the growth room for
149
another 4-6 months.
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Data analysis
Each of the treatments was performed three times. Statistical analyses were performed
153
according to the one-way analysis of variance (ANOVA) using SPSS software (version 17.0)
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to assess significant differences in the mean values of different treatments. Comparisons
155
between the mean values were assessed using Duncan’s multiple-range test (P < 0.05).
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RESULTS AND DISCUSSION
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Induction of callus from adventitious roots
Initiation of callus from adventitious root explants generally occurred after 3 weeks on
160
media supplemented with different combinations of growth regulators. The highest frequency
161
of callus induction was observed on the medium containing 0.5 mg/L 2,4-D and 0.3 mg/L
162
kinetin. The frequency of callus induction reduced dramatically as the concentration of 2,4-D
163
increased. Callus was not induced in the presence of 2 mg/L 2,4-D (Table 1). Similar results
164
were reported with the cultures of hairy roots of P. ginseng that 2,4-D at more than 3 mg/L
165
strongly suppressed callus induction [33]. When the segments of adventitious roots (Fig. 1A)
166
of P. ginseng were incubated in MS solid medium with 0.5 mg/L 2, 4-D and 0.3 mg/L kinetin,
167
callus was induced from the cut sides of the adventitious roots after 6 weeks of culture (Fig.
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169
three months, embryogenic callus was induced (Fig. 1C) and the embryogenic callus showed
170
high regenerative capacity and differentiated into somatic embryos and plantlets. Callus
171
induction and growth from adventitious root explants was dependent upon 2,4-D as
172
previously reported [22,24,27]. When embryogenic callus was transferred to MS medium
173
lacking kinetin, a small number of globular embryos formed after 3 weeks of culture (Fig. 1D,
174
E). Thus, it is essential to induce and maintain the embryogenic callus in the medium
175
supplemented with 2,4-D in combination with kinetin. Embryogenic callus has been
176
maintained in the dark for more than 2 years through 3-week subculture intervals on MS solid
177
medium with 0.5 mg/L 2,4-D and 0.3 mg/L kinetin.
178 179
Induction of somatic embryos
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The embryogenic callus grows better in a liquid medium than a solid medium (data not
181
shown). Therefore, we propagated the embryogenic callus in bioreactor to assess somatic
182
embryo development and plantlet conversion. When embryogenic callus was inoculated into
183
a 15 L airlift bioreactor containing 5 L MS liquid medium with 0.5 mg/L 2,4-D, the
184
embryogenic callus was propagated and a small number of globular shaped embryos were
185
also formed after 3 weeks of culture (Fig. 1D, E). The growth rate (final explant fresh
186
weight/initial explant fresh weight) was about 2.1. Embryogenic cell clumps proliferated in
187
bioreactor were transferred onto MS solid medium with different concentrations of 2,4-D (0,
188
0.5 or 1.0 mg/L) for embryogenesis. The frequency of somatic embryo formation was
189
significantly depended on the concentrations of 2,4-D (Table 2). The highest induction
190
frequency of somatic embryos was observed on the medium supplemented with 0.5 mg/L
191
2,4-D. The frequency of somatic embryo formation in wild-type and mutant cell line was
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193
in wild-type and mutant cell line, respectively. There was no significant difference in somatic
194
embryo formation frequency between wild-type and mutant cell line (Table 2). Globular
195
shaped somatic embryos formed on the surfaces of embryogenic callus (Fig. 1F, G).
196 197
Maturation and germination of somatic embryos
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These somatic embryos were transferred into 500 mL-Erlenmeyer flasks containing 200
199
mL of liquid MS medium supplemented with 0.5 mg/L 2,4-D and 3% sucrose (Fig. 1H) for
200
proliferation. The growth rate (final explant fresh weight/initial explant fresh weight) was
201
about 1.7. After 4 weeks of culture, the proliferated globular embryos were transferred to
202
petri dishes containing solid MS medium with various concentrations of GA3 and 3% sucrose.
203
At 5 mg/L GA3, most of the globular embryos turned green and increased in size and
204
developed into torpedo and cotyledonary stage embryos within one month. When the mature
205
somatic embryos were transferred to a fresh medium with the same composition, most of the
206
embryos germinated within 2 weeks of culture (Fig. 1I). Adventitious shoots were induced
207
from the mature somatic embryos. The optimal concentration of GA3 in germination medium
208
was 5 mg/L, yielding the highest germination frequency of 85%. Without GA3 treatment, the
209
germination frequency was lowest at 36%. Maturation and germination of embryos were
210
strongly influenced by the GA3 concentration (Table 3). This result suggests that GA3 is
211
required for maturation and germination of somatic embryos. Similar results were observed
212
in Eleutherococcus senticosus, that GA3 treatment was necessary to induce germination from
213
somatic embryos [34]. GA3 treatment is also commonly used for maturation and germination
214
of somatic embryos from P. ginseng [22,26,28-29], from P. quinquefolius [35] and from P.
215
japonicus [36].
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Development of plantlets and transplantation When shoots reached 0.5-1.0 cm in height on germination medium, the shoots were
219
transferred to elongation medium, 50 mL MS solid medium supplemented with 5 mg/L GA3
220
in 100×40 mm plastic petri dishes, for further growth of shoots. After about one month of
221
culture, the shoots developed to 3.0-4.0 cm in height, but most of the shoots had no visible
222
roots. The shoots without roots were excised and transferred to different rooting media, half
223
or one-third strength MS, or SH basal medium supplemented with 0.25 mg/L NAA or with
224
0.5% activated charcoal, in 75×130 mm glass bottles, one shoot per bottle. Adventitious roots
225
formed from the excised regions of the shoots. After 1 month, the rate of root formation from
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the shoots was examined (Table 4). As far as root quality is concerned, 1/3 SH medium with
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0.25 mg/L NAA and 1% sucrose showed the best result among the tested rooting media; the
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roots grew fast and thickened on the medium (Fig. 1J; Fig. 2A, B; Table 4). Although 1/3 SH
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medium with 2% sucrose and 0.5% activated charcoal was most effective in inducing roots,
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the roots grew well but weak (Table 4). The optimal medium for rooting is therefore 1/3 SH
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medium supplemented with 0.25 mg/L NAA and 1% sucrose among the tested rooting media
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in this study. In our comparative studies, SH medium was more effective than MS medium in
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root induction and proliferation. A very similar result was reported in American [35] and
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Korean ginsengs [30]. It was reported that the high level of ammonium nitrate in MS medium
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highly suppressed root development in carrot [37]. Choi et al. [5] reported that when the
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ammonium nitrate was omitted in MS medium, root growth of regenerated ginseng plants
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was enhanced. The concentration of ammonium nitrate in SH medium was about 8 times
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lower than in MS medium. It seems that the different concentrations of ammonium nitrate in
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SH and MS medium may result in the different root induction efficiency between the two
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basal medium. From these observations, we suggest that SH medium, especially 1/3 strength
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SH medium is suitable for root induction and growth of regenerated ginseng plants. Well-developed plantlets with both shoots and roots derived from adventitious roots were
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transferred to plastic pots (10×18 cm) containing an artificial soil mixture of peat moss,
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vermiculite and perlite (2:3:1 v/v) in a growth room (Fig. 2C). The survival rate of the
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plantlets was about 30% after 3 months of culture and new leaf began growing (Fig. 2D). The
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plants regenerated from both wild-type and mutant cell line acclimatized in the growth room
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(Fig. 3).
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In conclusion, we developed an efficient in vitro regeneration protocol for an important
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medicinal plant of P. ginseng. The protocol described here will allow a relatively rapid mass
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production of Korean wild ginseng plants. It takes 6-8 months from the callus induction of
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adventitious roots to the plantation of plants. In the present study, we also produced the
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regenerated plants from the mutant adventitious roots which were obtained by γ-irradiation.
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The combination of mutation technique by γ-irradiation and plant regeneration by tissue
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cultures may be an effective way to ginseng improvement. The protocol established in this
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study is currently being used for the genetic transformation of this species.
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ACKNOWLEDGEMENTS This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2009-0094059).
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35. Zhou S, Brown DCW. High efficiency plant production of North American ginseng via somatic embryogenesis from cotyledon explants. Plant Cell Rep 2006;25:166-173. 36. You XL, Han JY, Choi YE. Plant regeneration via direct somatic embryogenesis in Panax
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TABLES AND FIGURES
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Table 1. Effects of 2,4-D and kinetin on the frequency of callus formation from ginseng
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adventitious roots
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2, 4-D
Kinetin
Number of
Number of root explants
(mg/L)
(mg/L)
root explants
formed callus
0.5
0
30
4.3 ±1.0de
0.3
30
0.5
30
0
30
0.3
30
0.5 2
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9.1 ± 1.7de 7.2 ± 1.2bc
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16.2 ± 1.8a
0 0.3 0.5
30
2.3 ± 0.8ef
30
0.0 ± 0f
30
0.0 ± 0f
30
0.0 ± 0f
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5.4 ± 1.1cd
The data were collected after 6 weeks of culture.
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The results represent the means ± SEM of values obtained from three experiments.
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Different corresponding letters within a column are significant different at P<0.05 by
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Duncan’s multiple range test.
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Figure 1. Somatic embryogenesis and regeneration of plantlet from adventitious roots of
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Panax ginseng. (A) Adventitious roots derived from Korean wild ginseng root. (B) Callus
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induction from adventitious root explants. (C) Embryogenic callus derived from adventitious
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roots. (D) Proliferation of embryogenic callus in an airlift bioreactor. (E) Proliferated
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embryogenic cell clumps from bioreactor culture. (F) Somatic embryos formed on
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embryogenic callus. (G) Magnified image from (F) (scale bar = 2 mm). (H) Proliferation of
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somatic embryos in conical flasks. (I) Maturation and germination of somatic embryos on MS
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medium supplemented with 5 mg/L GA3. (J) Well-developed plantlet derived from somatic
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embryo (scale bar = 0.8 cm).
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Table 2. Effects of 2,4-D on somatic embryo formation from embryogenic callus of wild-type
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and mutant adventitious roots
Mutant
Frequency of somatic
Number of somatic
(mg/L)
embryo formation (%)
embryos per callus
0
5.3 ± 0.73b
10.0 ± 1.2b
0.5
15.3 ± 1.21a
1
2.5 ± 0.46c
0
5.8 ± 0.28b
0.5
14.7 ± 0.45a
1
2.2 ± 0.27c
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Wild-type
2,4-D
25.6 ± 2.3a 4.7 ± 0.3c
12.3 ± 1.9b 23.7 ± 0.6a
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Cell line
6.0 ± 1.4c
The data were collected after 6 weeks of culture.
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The results represent the means ± SEM of values obtained from three experiments.
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Different corresponding letters within a column are significant different at P<0.05 by
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Duncan’s multiple range test.
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Number of somatic
Germination frequency
(mg/L)
embryos inoculated
embryos germinated
(%)
0
150
53 ± 6c
36 ± 4c
1
150
60 ± 9c
40 ± 6c
3
150
66 ± 10bc
5
150
127 ± 7a
7
150
75 ± 6b
10
150
65 ± 5bc
45 ± 7bc 85 ± 5a
50 ± 4b
44 ± 3bc
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The data were collected after 6 weeks of culture on MS medium with 3% sucrose.
415
The results represent the means ± SEM of values obtained from three experiments.
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Different corresponding letters within a column are significant different at P<0.05 by
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Duncan’s multiple range test.
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Root frequency
No. of roots
Description on
shoots
(%)
per plant
root quality
1/2 MS 3% Sucrose
30
36
1.6
Grows slow, calluses
1/2 MS + 3% Sucrose + 0.5% charcoal
30
45
1.0
Grows slow
1/3 MS + 1% Sucrose + 0.25 mg/L NAA
30
58
1/2 SH + 3% Sucrose
30
71
1/2 SH + 2% Sucrose + 0.5% charcoal
30
62
1/3 SH + 2% Sucrose + 0.5% charcoal
30
80
1/3 SH + 1% Sucrose + 0.25 mg/L NAA
30
76
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Grows fast, thin
1.0
Grows slow
1.0
Grows slow, calluses
1.0
Grows fast, thin
1.2
Grows fast, strong
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Figure 2. Development of ginseng plant and transplantation into soil. (A) Well-developed
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plantlet on rooting medium, 1/3 SH basal medium supplemented with 0.25 mg/L NAA. (B)
430
Plant with a taproot just before transplantation into soil. (C) Regenerated plant hardened in
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soil. (D) New leaves (indicated by arrows) were produced from three-month old potted plant
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(scale bar = 0.8 cm).
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Figure 3. Transplantation of regenerated ginseng plants into soil. (A-E) Plants derived from
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wild-type adventitious roots. (F-J) Plants derived from mutant adventitious roots.
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