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Low dose gamma radiation increases the biomass and ginsenoside content of callus and adventitious root cultures of wild ginseng (Panax ginseng Mayer)
Industrial Crops & Products 130 (2019) 16–24
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Low dose gamma radiation increases the biomass and ginsenoside content of callus and adventitious root cultures of wild ginseng (Panax ginseng Mayer) Kim-Cuong Le, Thanh-Tam Ho, Kee-Yoeup Paek, So-Young Park
T
⁎
Department of Horticultural Science, Chungbuk National University, Cheongju, Chungbuk 28466, Republic of Korea
A R T I C LE I N FO
A B S T R A C T
Keywords: Adventitious roots Biomass Compound K Gamma irradiation Ginsenosides Mutagenesis
Mountain ginseng (Panax ginseng Mayer) is known as the most valuable herb around the world. Several biotechnological tools have been developed to enhance the production of secondary metabolites in medicinal plants, especially mountain ginseng. Mutagenesis is used to create desirable mutations at the genetic level. Among several energy sources used in mutant breeding programs, gamma rays are considered the most common and efficient tool for mutagenesis. Gamma rays are preferred over other energy sources because of convenient operation, short cycle, and high mutation frequency. In this study, gamma rays were used to generate mutant adventitious root lines of P. ginseng with high biomass and ginsenoside content. Callus and adventitious root cultures of P. ginseng, which were in culture short-term (1 year) and long-term (20 years), were irradiated with various doses of gamma rays. Exposure to higher doses of gamma rays caused necrosis in long-term explants. Four mutant lines of adventitious roots were identified with unique RAPD markers using eight primers. The genetic similarity among mutant lines ranged from 0.67 to 0.96. We also analyzed the total ginsenoside content and levels of 12 individual ginsenosides using HPLC analysis. The total ginsenoside content was 4.2-fold higher in mutant lines than in the control. The mutant line 1G-20-19 showed the highest ginsenoside content compared with the untreated control and other mutant lines. Overall, mutant lines of adventitious roots by gamma ray treatments in this study represent good candidates for commercial production of ginsenosides.
1. Introduction Plant secondary metabolites are important sources of medicines, agrochemicals, cosmetics, nutraceuticals, and food products. The quality and quantity of bioactive compounds in plants are considerably affected by biotic and abiotic factors (Murch and Saxena, 2006). The increasing demand for raw plant materials for the production of medicinal products has caused the destruction of natural habitats and a reduction in plant populations and genetic diversity, leading to species extinction (Dal Toso and Melandri, 2011). Plant biotechnology has become a reliable method for the production of secondary metabolites in important medicinal plants under controlled conditions (Kolewe et al., 2008; Baque et al., 2012; Gaosheng and Jingming, 2012; Dias et al., 2016). Among the different biotechnological techniques, adventitious root culture has gained importance because of the rapid growth rate of tissues and easy extraction of stable bioactive compounds (Hahn and Yu KW, 2003; Murthy et al., 2016). Recent advancements in plant
biotechnology have enabled the establishment of large-scale adventitious root cultures with high biomass using a bioreactor culture system (Choi et al., 2000; Sivakumar, 2006; Baque et al., 2012). However, pilot-scale production of secondary metabolites through in vitro plant culture is still challenging because of inconsistent materials and low metabolite yield during long-term subculture (Bogdanova et al., 2009; Figueiró A de et al., 2010; Sánchez-Rojo et al., 2015). The reduction in the quality of secondary metabolites during long-term in vitro culture may induce alterations in DNA methylation patterns (González et al., 2013; Machczyńska et al., 2014). Several strategies have been used for enhancing the production of secondary metabolites in plants. In the last decade, gamma rays have been used to develop new plant varieties with higher level of bioactive compounds (Jan et al., 2012; Jaisi et al., 2013). Gamma rays interact with atoms and molecules to stimulate the production of intracellular primary (%OH, H%) and secondary (H2O2, O%2−) free radicals, which play key roles in plant defense responses and accumulation of secondary metabolites (Wi et al., 2007; Esnault et al., 2010; Azeez et al., 2017). A
Abbreviations: CK, compound K; PPD, protopanaxadiol; PPT, protopanaxatriol; RAPD, random amplified polymorphic DNA ⁎ Corresponding author at: Department of Horticultural Science, Division of Animal, Horticulture and Food Sciences, Chungbuk National University, Cheongju 28466, Republic of Korea. E-mail address: [email protected] (S.-Y. Park). https://doi.org/10.1016/j.indcrop.2018.12.056 Received 10 October 2018; Received in revised form 17 December 2018; Accepted 17 December 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.
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higher level of different types of metabolites, including phenolics, flavonoids, terpenoids, and alkaloids, has been reported in many medicinal plants by γ-irradiation (Vardhan and Shukla, 2017). Wild ginseng (Panax ginseng Mayer) is an important herb and has been used as a traditional medicine and functional food in the Orient (Christensen, 2008; Zhang et al., 2011; Murthy et al., 2014). Over the last 20 years, adventitious root culture has been established from the callus derived from 100-yr-old ginseng in Chungbuk National University. During this time, the culture has been maintained via continuous subculture at 8 week intervals. However, the biomass yield and ginsenoside content of these cultures has decreased over time. Mutagenesis of P. ginseng can create novel lines with high productivity and ginsenoside content. Recently, mutant lines generated using gamma rays have been shown to exhibit 1.8-fold (Zhang et al., 2011) and 1.7fold (Kim et al., 2013) higher ginsenoside content than the wild type. In this study, the novel P. ginseng lines were developed with high biomass and ginsenoside content using gamma irradiation of adventitious root cultures.
Table 1 List of random amplified polymorphic DNA (RAPD) primers used in this study. Primer
Sequence (5′→3′)
A-05 A-08 A-09 A-10 A-13 A-15
AGGGGTCTTG GTGACGTAGG GGGTAACGCC GTGATCGCAG CAGCACCCAC TTCCGAACCC
bromide (CTAB) extraction method (Doyle 1987). The CTAB buffer and 0.2% β-mercaptoethanol were added to the homogenized samples and incubated at 65 °C for 30 min. Subsequently, chloroform:isoamyl alcohol (24:1) extraction solution was added, and DNA was precipitated with isopropanol. DNA pellets were washed with 70% ethanol and resuspended in 50 μL of sterilized water. DNA concentration was measured using a DS-11+ spectrophotometer (Denovix, Inc., USA).
2. Materials and methods 2.3.2. PCR amplification Amplification was performed using a C1000™ Thermal Cycler (CFX96 Touch™ Real-time PCR Detection System; Bio-Rad Laboratories, Inc., Singapore). Twenty oligonucleotides (Operon Inc., USA) were used for the initial round of PCR amplification, and six primers (A-05, A-08, A-09, A-10, A-13, and A-15) were subsequently selected for further amplifications (Table 1). PCRs were conducted in a total volume of 20 μL containing DNA template (10 ng·μL−1), 2× Primer Tag Premix (Genet Bio Inc., Korea), primers, and sterilized water. PCR products were separated, visualized, and scored on the QIAxcel (Qiagen) automatic capillary electrophoresis system.
2.1. Plant materials and culture conditions One-yr-old (short-term) and 20-yr-old (long-term) adventitious root cultures (AR1 and AR20, respectively) and callus (CS1 and CS20, respectively) of wild ginseng, available at the Chungbuk National University, were used in this study. The adventitious root cultures were maintained on quarter-strength Murashige and Skoog (¾MS) medium supplemented with 10.16 μM indole-3-butyric acid (IBA) and 5% sucrose. Callus was maintained on MS medium supplemented with 2.2 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4% sucrose. The pH of the medium was adjusted to 5.8 before autoclaving at 121 °C for 20 min. For Petri dish culture, 10 adventitious roots (1–1.5 cm long) and five callus clumps were cultured on solid MS medium (with 0.2% gelrite). For flask culture, fresh adventitious roots (0.2 g) were inoculated in a 100 mL flask containing 40 mL of liquid MS medium and incubated at 24 ± 1 °C on a shaker (100 rpm) in the dark. Adventitious root cultures and callus were subcultured every 40 and 30 days, respectively.
2.4. Preparation and extraction of mutant lines Root samples (0.5 g dry weight [DW]) of control and selected mutant lines were placed in 100 mL flasks containing 50 mL of 80% (v/v) ethanol. Samples in flasks were sonicated in an ultra-sonication bath (SD-D250 H; Mujigae Co., Korea) for 1 h at room temperature. Thereafter, extracts were filtered through filter paper (Whatman, UK) and collected in a round-bottom flask. The solvent was evaporated to dryness using a rotary evaporator (N-1000; Eyela, Japan) at 40 °C, and the residue was dissolved in 50 mL of distilled water. The aqueous solution was washed twice with 50 mL of ethyl ether, and the aqueous layer was extracted twice with 50 mL of water-saturated n-butanol. The n-butanol fraction was evaporated using a rotary evaporator at 50 °C. The sample was dissolved in 2 mL of methanol and filtered through a 0.2 μm syringe filter (Whatman) before being subjected to high performance liquid chromatography (HPLC).
2.2. Gamma irradiation treatment Root tips, 1–2 cm in length, were detached from short- and longterm adventitious root cultures (AR1 and AR20, respectively) of P. ginseng (10 explants per Petri dish with medium, five replications) and irradiated 24 h with six different doses of gamma rays (0, 20, 40, 60, 80, and 100 Gy; linear energy transfer [LET] = 0.2 keV·μm−1) using a 60Co source at the Korea Atomic Energy Research Institute. Callus (CS1 and CS20) cultures were treated with 0, 20, 30, 50, 75, and 100 Gy gamma rays (LET = 0.2 keV·μm−1) using the same 60Co source. After irradiation, both roots and callus were immediately transferred to fresh ¾MS medium supplemented with 10.16 μM IBA and 5% sucrose to induce the development of adventitious roots. The lethal dose 50 value (LD50) is represented the dose which give 50% necrosis and non-response adventitious roots after 30 days of gamma ray treatments. The root lines inducing efficiency were estimated by measuring the fresh and dry weight, lateral root number, length and diameter after 40 days of culture.
2.5. HPLC analysis of ginsenosides The extracted samples were analyzed via HPLC using the Waters 2695 separation module equipped with a 2996 photodiode array detector on a Fortis C18 column (φ 5 μm, 150 × 4.6 mm; UK). The mobile phase comprised acetonitrile (solvent A) and water (solvent B). Elution was performed at a constant flow rate of 0.6 mL·min−1 using the following gradient: 0 min, 18% A, 82% B; 0–42 min, 24% A, 76% B; 42–46 min, 29% A, 71% B; 46–75 min, 40% A, 60% B; 75–100 min, 65% A, 35% B; 100–135 min, 85% A, 15% B; and 135–150 min, 85% A, 15% B. Ginsenosides were detected at 203 nm. The sample injection volume was 20 μL, and the temperature of the column was maintained at 35 °C. Both protopanaxadiol (PPD)-type (Rb1, Rb2, Rb3, Rc, Rd, Rg3, Rh2, and CK) and protopanaxatriol (PPT)-type (Re, Rf, Rg1, and Rh1) ginsenosides (ChromaDex, USA) were used as standards. The ginsenoside content was calculated as follows:
2.3. Genotyping using random amplified polymorphic DNA (RAPD) marker analysis 2.3.1. DNA isolation Fresh 6-wk-old adventitious roots (0.1 g) were harvested from selected putative mutant lines and control (non-irradiated) tissues and homogenized using a Tissuelyser II (Qiagen, Germany). Genomic DNA was isolated from root samples using the cetyl trimethyl ammonium 17
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Ginsenoside content (mg·g−1 DW) = ginsenoside concentration (mg·L−1) × sample volume (L) / root DW (g)
CS20 (data not shown). Therefore, the 20 Gy dose was considered as optimal for the selection of mutant lines in ginseng. In this study, four mutant root lines (1G-20-12, 1G-20-16, 1G-20-19, and 1G-20-20) were selected from AR1 culture; these lines showed the highest growth rate at 20 Gy, high branching, and vigorous growth after 40 days of culture on solid MS medium (data not shown).
Total ginsenoside content was calculated as the sum of the 12 individual ginsenosides. Ginsenoside productivity was calculated as follows: Ginsenoside productivity (mg·L−1) = [Total ginsenoside content (mg·g−1 DW) × harvested root DW (g)] / volume of culture medium (L)
3.3. Identification of mutants using RAPD markers To identify DNA polymorphisms induced by gamma irradiation in mutant adventitious root lines, we used RAPD markers. Ten primers were screened in a preliminary analysis, of which four primers showed monomorphic bands. Among the remaining six primers, four primers (A5, A8, A9, and A10) showed highly polymorphic banding patterns, ranging from 300 to 3000 bp (Fig. 4A). To evaluate the genetic variation induced by gamma irradiation, a binary matrix of genetic distances was calculated based on the results of RAPD analysis (Table 2). RAPD data were used to create a dendrogram, which showed two distinct clusters. One of these clusters included two mutant lines (1G-20-20 and 1G-20-19) and the control, while the other cluster comprised 1G-20-12 and 1G-20-16 mutants, indicating higher distinctness from the other root lines (Fig. 4B).
2.6. Statistical analysis The presence or absence of DNA bands on the gel following PCR amplification using random amplified polymorphic DNA (RAPD) primers was scored as 1 or 0, respectively. These scores were entered into a binary matrix and analyzed using NTSYSpc 2.1. Genetic similarities among mutant lines and the control were calculated using Jaccard’s coefficient, and a dendrogram was established using unweighted pair group method with arithmetic mean (UPGMA). Data were analyzed using SPSS version 16.0 (SPSS Inc., USA). Where a significant difference (P < 0.05) was observed for a measured parameter, means were separated using Duncan’s multiple range test at 5% level of significance.
3.4. Growth characteristics of mutant root lines 3. Results The growth characteristics of the four mutant root lines were determined after 40 days of culture in flasks using liquid MS medium (Figs. 4C and 5 ). The selected mutant root lines showed more vigorous growth than the control. However, biomass production showed no significant difference among the mutant lines (Fig. 5A). The length of lateral roots was similar among all mutant root lines and the control, whereas the number of secondary roots was higher in mutant lines than in the control (Fig. 5B, C). In addition, the mutant line 1G-20-19 showed the longest lateral roots. The diameter of lateral roots was higher in the mutant lines 1G-20-19 and 1G-20-20 than in the other mutant root lines and the control (Fig. 5D).
3.1. Effect of 60Co gamma irradiation on the survival of adventitious root and callus in ginseng Owing to long-term maintenance, the survival rate of CS20 was significantly lower than that of the short-term callus (CS1) after treatment with different doses of gamma rays, and CS20 was more sensitive to gamma rays than CS1 (Figs. 1A and 2 ). The survival rate of both short- and long-term callus was the highest at 20 Gy (100% and 75%, respectively). At 30 and 50 Gy, the survival rate of CS1 was maintained at 100% but that of CS20 was reduced to 50% (Fig. 2). At 75 and 100 Gy gamma rays, the survival rate of CS20 was dramatically reduced, whereas that of CS1 declined only slightly (Fig. 2). These data suggest that the survival rate of ginseng callus is unaffected at 20 Gy gamma rays but inhibited at 30 Gy for CS20 and 75 Gy for CS1. The sensitivity of adventitious roots of wild ginseng to gamma rays was evaluated by comparing the lethal dose of gamma rays between long-term and short-term root cultures (Figs. 1B and 3 ). Highly significant differences were recorded among the different doses of gamma rays (Fig. 3). The rate of lethal rate of adventitious roots in both AR1 and AR20 cultures continued to increase with the increase in gamma dosage after irradiation (Fig. 3). Additionally, the AR20 culture was more sensitive to gamma irradiation than AR1 (Fig. 3). The lethal dose at which 50% of the cultures survived (LD50) compared with the control was 52.3 Gy for AR1 and 23.7 Gy for AR20, as shown by the best fit and broken line (Fig. 3). The frequency of genetic mutation and necrosis increased with the increase in the dosage of gamma irradiation (Fig. 3).
3.5. HPLC analysis of ginsenoside content The total ginsenoside content, ginsenoside productivity, and the content of 12 individual ginsenoside compounds (Rb1, Rb2, Rb3, Rc, Rd, Re, Rf, Rg1, Rg2, Rg3, Rh2, and CK) were compared among the four mutant root lines and the control (Table 3, Figs. 6 and 7). The total ginsenoside content was dramatically enhanced in the four mutant root lines compared with the control; the content of PPD- and PPT-type ginsenosides in mutant root lines was 2.3–4.2-fold and 6.2–9.0-fold higher, respectively, than in the control (Table 3). Among the PPD-type ginsenosides, Rg3 in control, 1G-20-12, and 1G-20-20, and Rh2 in 1G20-16, were undetected by HPLC (Fig. 6A). All four mutant lines showed enrichment of Rb2; the level of Rb2 in the mutant lines was 42.2–90.4-fold higher than in the control. Additionally, the ginsenoside Rb1 in 1G-20-16, 1G-20-19, and 1G-20-20 lines was enhanced by 191.9-, 280.8-, and 239.4-fold, respectively, compared with the control (Fig. 6A). Results of HPLC analysis also showed the enrichment of PPTtype ginsenosides in mutant roots compared with the control (Fig. 6B). The ginsenoside Re in mutant roots was enhanced by 79.2–136.3-fold compared with the control; the highest level of Re was identified in the mutant line 1G-20-19. Levels of Rg1 and Rg2 were the highest in mutant lines 1G-20-16 and 1G-20-12, respectively (Fig. 6B). Overall, the ginsenoside production in all four mutant lines was higher than that in the control.
3.2. Formation of adventitious roots from callus and AR cultures and selection of mutant adventitious root lines After gamma irradiation, callus was transferred to adventitious root induction medium for 30 days. The rate of adventitious root formation from both CS1 and CS20 was less than that from AR1 and AR20 cultures, and the roots were shorter, smaller, and slow growing. Each lateral root detached from AR1 and AR20 cultures and irradiated with different doses was considered as one line. The AR1 culture showed more lateral root formation than AR20. A total of 138 lines were isolated from AR1 and AR20 cultures treated with different doses of gamma rays. The frequency of adventitious root formation was highest in AR1 treated with 20 Gy gamma rays compared with AR20, CS1, and
4. Discussion The positive response of callus and adventitious root cultures to low doses of gamma irradiation is consistent that low doses of irradiation 18
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Fig. 1. Effect of different doses of gamma ray in 24 h of short- and long-term callus (A) (CS1 and CS20, respectively) and adventitious root (B) (AR1 and AR20, respectively) Panax ginseng after 4 weeks of treatment (scale bar = 1 cm).
Fig. 3. Determination of LD50 value of gamma rays at different doses for short(AR1) and long-term (AR20) adventitious root cultures of P. ginseng after 30 days of culture. Data represent mean ± SEM of five replications. Dashed lines indicate the LD50 values. AR1, 1-yr-old adventitious root culture; AR20, 20-yrold adventitious root culture.
Fig. 2. Effect of different doses of gamma rays for 24 h on the survival rate of short- (CS1) and long-term callus (CS20) of Panax ginseng at 30 days after treatment. Data represent mean ± standard error of mean (SEM) of five replications. Different letters indicate statistically significant differences (P < 0.05), according to Duncan’s multiple range test. CS1, 1-yr-old callus; CS20, 20yr-old callus.
hormone signaling networks in cells, and uptake of mineral nutrients (Freidman, 1985; Ghiorghita et al., 1985; Antonov et al., 1989; AlOudat, 1990; Kim et al., 2004; Ling et al., 2008). The inhibition of growth caused by higher dosage of gamma rays may be related to the restriction of cell cycle at the G2/M phase and increase in genetic instability (Preuss and Britt, 2003). The survival rate and LD50 values are used to determine the irradiation dosage that is most likely to induce mutations (Mba et al., 2010). While high doses of gamma rays increase the mutation frequency, the lethal rate of mutants also increases; thus, the mutants generated using high doses of gamma rays are less valuable and have lower potential for commercial use (Brunner, 1995). The
have stimulatory effects on the growth of plant tissues (Fowler and MacQueen, 1972; Charbaji and Nabulsi, 1999; Kovalchuk et al., 2007). The lower survival rate and LD50 values of CS20 and AR20 than of CS1 and AR1 indicate that long-term cultures are more radiosensitive than short-term cultures. The enhanced survival rate of ginseng callus and adventitious roots exposed to low doses of gamma irradiation may result from the increased biosynthesis of amino acids (e.g., lysine and phenylalanine), modification of enzyme activities (e.g., catalases and peroxidases), increase in primary metabolite processes, alteration of 19
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Fig. 4. Characterization of mutant adventitious root lines of P. ginseng. (A) QIAxcel gel image of PCR products amplified using random polymorphic amplified DNA (RAPD) primers. (B) Dendrogram of mutant adventitious root lines based on RAPD markers. (C) Morphology of mutant lines after 40 days of culture (scale bar = 1 cm).
gamma irradiation should be identified that induces sufficient mutations while minimizing the negative effects on genome integrity. The physiological features of plant tissues are an important indicator of genetic variation and facilitate the selection of high quality genotypes with enhanced productivity. In the present study, 20 Gy treatment generated mutant adventitious root lines with high biomass, of which four mutant lines, exhibiting a wide range of variability in the number, length, and diameter of lateral roots, were selected (Fig. 5). Physiological characteristics have also been used previously for efficient screening of valuable mutants (de Ronde et al., 2009; Verma et al., 2010). The enhancement of biomass yield and alteration of physiological characteristics in mutant root lines are linked to the level, transport, and signaling of endogenous hormones (George et al., 2008; Atak et al., 2011). RAPD analysis is used for the detection of DNA polymorphisms caused by DNA breakage and adduction, point mutations, and large rearrangements. RAPD analysis has been used for mutant detection in many plant species, including Rosa indica (Chakrabarty and Datta, 2010), Rhododendron (Atak et al., 2011), Jatropha curcas
Table 2 Analysis of genetic similarity among four mutant adventitious root lines of Panax ginseng generated using gamma rays and the untreated control.
Control 1G-20-12 1G-20-16 1G-20-19 1G-20-20 a
Control
1G-20-12
1G-20-16
1G-20-19
1G-20-20
1.00a 0.60 0.69 0.77 0.80
1.00 0.86 0.71 0.63
1.00 0.80 0.71
1.00 0.80
1.00
Binary matrix was analyzed using NTSYSpc 2.1 software.
studies as the cellular level on plants irradiated with gamma rays also showed that high-dose irradiation inhibited the growth remarkably (Wi et al., 2007; Kovács and Keresztes, 2002). The cell membranes in Arabidopsis, particularly thylakoids were heavily swollen and disrupt; the structure of chloroplast was altered; and the concentration of H2O2 was also enhanced in plasma membranes and middle lamellae under highdose gamma radiation (Wi et al., 2007). Therefore, an optimal dose of 20
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Fig. 5. Characterization of growth phenotypes of putative mutant roots of P. ginseng induced by gamma irradiation of adventitious roots after 40 days of culture in Petri dishes. (A) Fresh weight and dry weight; (B) number of lateral roots per explant; (C) lateral root length; (D) root diameter. Data represent mean ± SEM of 5 replications. Different letters indicate statistically significant differences (P < 0.05), according to Duncan’s multiple range test (n = 5).
be caused by alterations in oligonucleotide priming sites, large deletions, and homologous recombination due to mutations (Atienzar and Jha, 2006; Laskar et al., 2018). The loss of bands may be attributed to DNA damage, while the appearance of new bands may be attributed to mutations. The majority of studies on mutation induction have focused on the development of novel lines with higher yield and secondary metabolite accumulation, especially in medicinal plants. In the present study, ginsenoside levels in mutant roots derived from gamma irradiation treatment were higher than in control roots (Table 3). Both PPD- and PPT-type ginsenosides showed higher accumulation in mutant roots, resulting in higher total ginsenoside content and productivity in mutant roots than in the control (Table 3 and Fig. 6). Previously, Kim et al. (2009) showed that mutant ginseng roots accumulate 2.5-fold higher ginsenoside content than wild-type ginseng. The HPLC analysis of mutant lines demonstrated three unknow ginsenoside types. The peak areas of unidentified ginsenoside candidates were higher from 46 to 65% than Rg1, which was the highest content in both mutant and wildtype ginseng (Kim et al., 2009). In this study, the total ginsenoside content of 1G-20-12, 1G-20-16, 1G-20-19, and 1G-20-20 mutant roots
(Dhakshanamoorthy et al., 2011; Dhillon et al., 2014), Acorus calamus (Lee and Han, 2014), Cuminum cyminum (Salarizadeh and Kavousi, 2015), Typhonium flagelliforme (Sianipar and Maarisit, 2015), and Lens culinaris (Laskar et al., 2018). Polymorphisms in genomic DNA are used to evaluate diversity and similarity among samples based on the presence or absence of DNA bands amplified using RAPD primers (Lee and Han, 2014; Ercan, 2015; Salarizadeh and Kavousi, 2015). In the present study, analysis of gamma ray-induced DNA polymorphism using RAPD markers showed differences in the selected adventitious root lines of P. ginseng. The dendrogram results implied that the four selected root lines carried mutations, which distinguished them from the control (Fig. 4). Among the four mutant lines, 1G-20-12 and 1G-20-16 were the most distinct from the control. The number and molecular weight of DNA bands in irradiated mutant root lines of P. ginseng were altered compared with the control. This implies that gamma rays induced severe DNA damage in the majority of the cells in adventitious roots of ginseng. The absence of normal bands might be related to single and double strand DNA breaks, base modifications, DNA-protein crosslinking, point mutations, and chromosomal rearrangements due to gamma irradiation. The presence of extra bands in RAPD profiles may
Table 3 Changes in total ginsenoside content and ginsenoside productivity of mutant Panax ginseng roots by HPLC analysis. Line
Protopanaxadiol (PPD)(mg·g−1 DW)
Protopanaxatriol (PPT)(mg·g−1 DW)
PPD/PPT
Total ginsenoside content (mg·g−1 DW)
Ginsenoside productivity (mg·L−1)
Control 1G-20-12 1G-20-16 1G-20-19 1G-20-20
1.30cz 2.54b 2.05bc 4.58a 3.09b
0.23b 2.09a 1.45a 1.88a 1.48a
5.99a 1.22b 1.41b 2.43b 2.24b
1.53c 4.62b 3.51b 6.46a 4.57b
6.63b 48.61a 51.82a 63.22a 50.49a
z
Different letters within a column indicate significant difference at P < 0.05 according to Duncan’s multiple range test (n = 3). 21
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Fig. 6. Content of individual ginsenosides in mutant adventitious root lines of P. ginseng after 40 days of culture. (A) Protopanaxadiol-type ginsenosides; (B) protopanaxatriol-type ginsenosides. Data represent mean ± SEM of 3 replications. Different letters indicate statistically significant differences (P < 0.05), according to Duncan’s multiple range test (n = 3).
upregulated to gamma rays resulted in the enhancement of ginsenoside content of mutant roots (Kim et al., 2013). Our data showed that the ginsenoside profiles of adventitious roots are affected by the activity of different enzymes in the ginsenoside biosynthesis pathway, the phenotype of adventitious root lines, and culture conditions. Similar results were reported in previous studies (Bonfill et al., 2002; Kim et al., 2009, 2013; Zhang et al., 2011).
were 3.0-, 2.3-, 4.2-, and 3.0-fold higher than that of the control, respectively (Table 3). The content of individual ginsenosides in all four mutant root lines was also higher than that in the control (Fig. 6). The results of Bonfill et al. (2002) imply enhanced biosynthesis of PPD-type ginsenosides due to the presence of auxins, such as IBA. Moreover, it is possible that genes involved in the biosynthesis of PPD-type ginsenosides may be mutated, leading to enhanced levels of these ginsenosides. Additionally, higher content of PPT-type ginsenosides in mutant ginseng roots may be caused by alterations in the activity of principal enzymes involved in their biosynthesis. Kim et al. (2013) showed that the transcription levels of P. ginseng squalene epoxidase and squalene synthase genes were remarkably enhanced by gamma irradiation of ginseng root culture. Dammarenediol synthase, phytosterol synthase, oleanane-type synthase genes were also reported significantly
5. Conclusions Long-term callus and adventitious root cultures of P. ginseng were more sensitive to gamma ray exposure than short-term cultures. Based on DNA fingerprints of adventitious roots, the mutant lines were clustered into two groups; 1G-20-12 and 1G-20-16 were the most diverse
Fig. 7. Chromatogram of 12 standards mixture (A), control (B) and mutant line 1G-20-19 (C) of ginsenoside compounds in Panax ginseng. 22
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from the control. The four mutant lines generated using gamma irradiation were characterized by high biomass yield and efficient production of ginsenosides. The mutant root line 1G-20-19 showed the highest biomass, ginsenoside content, and ginsenoside productivity compared with the control and other mutant lines. Overall, our data suggest that gamma irradiation is a powerful method for increasing the production of valuable secondary metabolites in plants, especially ginsenosides.
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Author contributions Kim-Cuong Le acquired data and wrote the manuscript, and ThanhTam Ho participated in analysis of HPLC. Kee-Yoeup Paek participated in interpretation of data and revision for important intellectual content. So-Young Park conceptualized and designed the study. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. Funding This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through Advanced Production Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (grant number 315013-4). Conflict of interest The authors declare that they have no conflicts of interest. Acknowledgments We thank Dr. Dong-Sub Kim and Mr. Jin-Baek Kim from Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute (KAERI), for technical assistance. References Al-Oudat, M., 1990. Effect of low dose gamma irradiation on onion yield. Ann. Biol 6, 61–67. Antonov, M., Velichov, P., Tsonev, T.S., Angelov, M., 1989. Effect of gamma and laser irradiation on maize seeds and plants. ESNA, XXth Annual Meeting p 44. Atak, Ç, Celik, O., Acik, L., 2011. Genetic analysis of Rhododendron mutants using random amplified polymorphic DNA (RAPD). Pak. J Bot 43, 1173–1182. Atienzar, F.A., Jha, A.N., 2006. The random amplified polymorphic DNA (RAPD) assay and related techniques applied to genotoxicity and carcinogenesis studies: a critical review. Mutat. Res. Mutat. Res. 613, 76–102. Azeez, H., Ibrahim, K., Pop, R., Pamfil, D., Hârţa, M., Bobiș, O., 2017. Changes induced by gamma ray irradiation on biomass production and secondary metabolites accumulation in Hypericum triquetrifolium Turra callus cultures. Ind Crops Prod. 108, 183–189. Baque, M.A., Moh, S.H., Lee, E.J., Zhong, J.J., Paek, K.Y., 2012. Production of biomass and useful compounds from adventitious roots of high-value added medicinal plants using bioreactor. Biotechnol Adv. 30, 1255–1267. Bogdanova, Y., Stoeva, T., Yanev, S., Pandova, B., Molle, E., Burrus, M., Stanilova, M., 2009. Influence of plant origin on propagation capacity and alkaloid biosynthesis during long-term in vitro cultivation of Leucojum aestivum L. In Vitro Cell. Dev. Biol. 45, 458–465. Bonfill, M., Cusidó, R.M., Palazón, J., Piñol, T., Morales, C., 2002. Influence of auxins on organogenesis and ginsenoside production in Panax ginseng calluses. Plant Cell Tissue Organ Cult. 68, 73–78. Brunner, H., 1995. Radiation induced mutations for plant selection. Appl. Radiat. Isot. 46, 589–594. Chakrabarty, D., Datta, S.K., 2010. Application of RAPD markers for characterization of γray-induced rose mutants and assessment of genetic diversity. Plant. Biotechnol Rep. 4, 237–242. Charbaji, T., Nabulsi, I., 1999. Effect of low doses of gamma irradiation on in vitro growth of grapevine. Plant Cell Tissue Organ Cult. 57, 129–132. Choi, S.M., Son, S.H., Yun, S.R., Kwon, O.W., Seon, J.H., Paek, K.Y., 2000. Pilot-scale culture of adventitious roots of ginseng in a bioreactor system. Plant Cell Tissue
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Microbiol. Biotechnol. 98, 6243–6254. Murthy, H.N., Dandin, V.S., Paek, K.Y., 2016. Tools for biotechnological production of useful phytochemicals from adventitious root cultures. Phytochem. Rev. 15, 129–145. Preuss, S.B., Britt, A.B., 2003. A DNA-damage-induced cell cycle checkpoint in Arabidopsis. Genetics 164, 323–334. Salarizadeh, S., Kavousi, H.R., 2015. Application of random amplified polymorphic DNA (RAPD) to detect the genotoxic effect of cadmium on tow iranian ecotypes of cumin (Cuminum cyminum). J Cell. Mol. Res. 7, 38–46. Sánchez-Rojo, S., Cerda-García-Rojas, C.M., Esparza-García, F., Plasencia, J., PoggiVaraldo, H.M., Ponce-Noyola, T., Ramos-Valdivia, A.C., 2015. Long-term response on growth, antioxidant enzymes, and secondary metabolites in salicylic acid pre-treated uncaria tomentosa microplants. Biotechnol. Lett. 37, 2489–2496. Sianipar, N.F., Maarisit, W., 2015. Detection of gamma-irradiated mutant of rodent tuber (Typhonium flagelliforme lodd.) in vitro culture by RAPD molecular marker. Procedia Chem. 14, 285–294.
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Low dose gamma radiation increases the biomass and ginsenoside content of callus and adventitious root cultures of wild ginseng (Panax ginseng Mayer) Kim-Cuong Le, Thanh-Tam Ho, Kee-Yoeup Paek, So-Young Park
T
⁎
Department of Horticultural Science, Chungbuk National University, Cheongju, Chungbuk 28466, Republic of Korea
A R T I C LE I N FO
A B S T R A C T
Keywords: Adventitious roots Biomass Compound K Gamma irradiation Ginsenosides Mutagenesis
Mountain ginseng (Panax ginseng Mayer) is known as the most valuable herb around the world. Several biotechnological tools have been developed to enhance the production of secondary metabolites in medicinal plants, especially mountain ginseng. Mutagenesis is used to create desirable mutations at the genetic level. Among several energy sources used in mutant breeding programs, gamma rays are considered the most common and efficient tool for mutagenesis. Gamma rays are preferred over other energy sources because of convenient operation, short cycle, and high mutation frequency. In this study, gamma rays were used to generate mutant adventitious root lines of P. ginseng with high biomass and ginsenoside content. Callus and adventitious root cultures of P. ginseng, which were in culture short-term (1 year) and long-term (20 years), were irradiated with various doses of gamma rays. Exposure to higher doses of gamma rays caused necrosis in long-term explants. Four mutant lines of adventitious roots were identified with unique RAPD markers using eight primers. The genetic similarity among mutant lines ranged from 0.67 to 0.96. We also analyzed the total ginsenoside content and levels of 12 individual ginsenosides using HPLC analysis. The total ginsenoside content was 4.2-fold higher in mutant lines than in the control. The mutant line 1G-20-19 showed the highest ginsenoside content compared with the untreated control and other mutant lines. Overall, mutant lines of adventitious roots by gamma ray treatments in this study represent good candidates for commercial production of ginsenosides.
1. Introduction Plant secondary metabolites are important sources of medicines, agrochemicals, cosmetics, nutraceuticals, and food products. The quality and quantity of bioactive compounds in plants are considerably affected by biotic and abiotic factors (Murch and Saxena, 2006). The increasing demand for raw plant materials for the production of medicinal products has caused the destruction of natural habitats and a reduction in plant populations and genetic diversity, leading to species extinction (Dal Toso and Melandri, 2011). Plant biotechnology has become a reliable method for the production of secondary metabolites in important medicinal plants under controlled conditions (Kolewe et al., 2008; Baque et al., 2012; Gaosheng and Jingming, 2012; Dias et al., 2016). Among the different biotechnological techniques, adventitious root culture has gained importance because of the rapid growth rate of tissues and easy extraction of stable bioactive compounds (Hahn and Yu KW, 2003; Murthy et al., 2016). Recent advancements in plant
biotechnology have enabled the establishment of large-scale adventitious root cultures with high biomass using a bioreactor culture system (Choi et al., 2000; Sivakumar, 2006; Baque et al., 2012). However, pilot-scale production of secondary metabolites through in vitro plant culture is still challenging because of inconsistent materials and low metabolite yield during long-term subculture (Bogdanova et al., 2009; Figueiró A de et al., 2010; Sánchez-Rojo et al., 2015). The reduction in the quality of secondary metabolites during long-term in vitro culture may induce alterations in DNA methylation patterns (González et al., 2013; Machczyńska et al., 2014). Several strategies have been used for enhancing the production of secondary metabolites in plants. In the last decade, gamma rays have been used to develop new plant varieties with higher level of bioactive compounds (Jan et al., 2012; Jaisi et al., 2013). Gamma rays interact with atoms and molecules to stimulate the production of intracellular primary (%OH, H%) and secondary (H2O2, O%2−) free radicals, which play key roles in plant defense responses and accumulation of secondary metabolites (Wi et al., 2007; Esnault et al., 2010; Azeez et al., 2017). A
Abbreviations: CK, compound K; PPD, protopanaxadiol; PPT, protopanaxatriol; RAPD, random amplified polymorphic DNA ⁎ Corresponding author at: Department of Horticultural Science, Division of Animal, Horticulture and Food Sciences, Chungbuk National University, Cheongju 28466, Republic of Korea. E-mail address: [email protected] (S.-Y. Park). https://doi.org/10.1016/j.indcrop.2018.12.056 Received 10 October 2018; Received in revised form 17 December 2018; Accepted 17 December 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.
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higher level of different types of metabolites, including phenolics, flavonoids, terpenoids, and alkaloids, has been reported in many medicinal plants by γ-irradiation (Vardhan and Shukla, 2017). Wild ginseng (Panax ginseng Mayer) is an important herb and has been used as a traditional medicine and functional food in the Orient (Christensen, 2008; Zhang et al., 2011; Murthy et al., 2014). Over the last 20 years, adventitious root culture has been established from the callus derived from 100-yr-old ginseng in Chungbuk National University. During this time, the culture has been maintained via continuous subculture at 8 week intervals. However, the biomass yield and ginsenoside content of these cultures has decreased over time. Mutagenesis of P. ginseng can create novel lines with high productivity and ginsenoside content. Recently, mutant lines generated using gamma rays have been shown to exhibit 1.8-fold (Zhang et al., 2011) and 1.7fold (Kim et al., 2013) higher ginsenoside content than the wild type. In this study, the novel P. ginseng lines were developed with high biomass and ginsenoside content using gamma irradiation of adventitious root cultures.
Table 1 List of random amplified polymorphic DNA (RAPD) primers used in this study. Primer
Sequence (5′→3′)
A-05 A-08 A-09 A-10 A-13 A-15
AGGGGTCTTG GTGACGTAGG GGGTAACGCC GTGATCGCAG CAGCACCCAC TTCCGAACCC
bromide (CTAB) extraction method (Doyle 1987). The CTAB buffer and 0.2% β-mercaptoethanol were added to the homogenized samples and incubated at 65 °C for 30 min. Subsequently, chloroform:isoamyl alcohol (24:1) extraction solution was added, and DNA was precipitated with isopropanol. DNA pellets were washed with 70% ethanol and resuspended in 50 μL of sterilized water. DNA concentration was measured using a DS-11+ spectrophotometer (Denovix, Inc., USA).
2. Materials and methods 2.3.2. PCR amplification Amplification was performed using a C1000™ Thermal Cycler (CFX96 Touch™ Real-time PCR Detection System; Bio-Rad Laboratories, Inc., Singapore). Twenty oligonucleotides (Operon Inc., USA) were used for the initial round of PCR amplification, and six primers (A-05, A-08, A-09, A-10, A-13, and A-15) were subsequently selected for further amplifications (Table 1). PCRs were conducted in a total volume of 20 μL containing DNA template (10 ng·μL−1), 2× Primer Tag Premix (Genet Bio Inc., Korea), primers, and sterilized water. PCR products were separated, visualized, and scored on the QIAxcel (Qiagen) automatic capillary electrophoresis system.
2.1. Plant materials and culture conditions One-yr-old (short-term) and 20-yr-old (long-term) adventitious root cultures (AR1 and AR20, respectively) and callus (CS1 and CS20, respectively) of wild ginseng, available at the Chungbuk National University, were used in this study. The adventitious root cultures were maintained on quarter-strength Murashige and Skoog (¾MS) medium supplemented with 10.16 μM indole-3-butyric acid (IBA) and 5% sucrose. Callus was maintained on MS medium supplemented with 2.2 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4% sucrose. The pH of the medium was adjusted to 5.8 before autoclaving at 121 °C for 20 min. For Petri dish culture, 10 adventitious roots (1–1.5 cm long) and five callus clumps were cultured on solid MS medium (with 0.2% gelrite). For flask culture, fresh adventitious roots (0.2 g) were inoculated in a 100 mL flask containing 40 mL of liquid MS medium and incubated at 24 ± 1 °C on a shaker (100 rpm) in the dark. Adventitious root cultures and callus were subcultured every 40 and 30 days, respectively.
2.4. Preparation and extraction of mutant lines Root samples (0.5 g dry weight [DW]) of control and selected mutant lines were placed in 100 mL flasks containing 50 mL of 80% (v/v) ethanol. Samples in flasks were sonicated in an ultra-sonication bath (SD-D250 H; Mujigae Co., Korea) for 1 h at room temperature. Thereafter, extracts were filtered through filter paper (Whatman, UK) and collected in a round-bottom flask. The solvent was evaporated to dryness using a rotary evaporator (N-1000; Eyela, Japan) at 40 °C, and the residue was dissolved in 50 mL of distilled water. The aqueous solution was washed twice with 50 mL of ethyl ether, and the aqueous layer was extracted twice with 50 mL of water-saturated n-butanol. The n-butanol fraction was evaporated using a rotary evaporator at 50 °C. The sample was dissolved in 2 mL of methanol and filtered through a 0.2 μm syringe filter (Whatman) before being subjected to high performance liquid chromatography (HPLC).
2.2. Gamma irradiation treatment Root tips, 1–2 cm in length, were detached from short- and longterm adventitious root cultures (AR1 and AR20, respectively) of P. ginseng (10 explants per Petri dish with medium, five replications) and irradiated 24 h with six different doses of gamma rays (0, 20, 40, 60, 80, and 100 Gy; linear energy transfer [LET] = 0.2 keV·μm−1) using a 60Co source at the Korea Atomic Energy Research Institute. Callus (CS1 and CS20) cultures were treated with 0, 20, 30, 50, 75, and 100 Gy gamma rays (LET = 0.2 keV·μm−1) using the same 60Co source. After irradiation, both roots and callus were immediately transferred to fresh ¾MS medium supplemented with 10.16 μM IBA and 5% sucrose to induce the development of adventitious roots. The lethal dose 50 value (LD50) is represented the dose which give 50% necrosis and non-response adventitious roots after 30 days of gamma ray treatments. The root lines inducing efficiency were estimated by measuring the fresh and dry weight, lateral root number, length and diameter after 40 days of culture.
2.5. HPLC analysis of ginsenosides The extracted samples were analyzed via HPLC using the Waters 2695 separation module equipped with a 2996 photodiode array detector on a Fortis C18 column (φ 5 μm, 150 × 4.6 mm; UK). The mobile phase comprised acetonitrile (solvent A) and water (solvent B). Elution was performed at a constant flow rate of 0.6 mL·min−1 using the following gradient: 0 min, 18% A, 82% B; 0–42 min, 24% A, 76% B; 42–46 min, 29% A, 71% B; 46–75 min, 40% A, 60% B; 75–100 min, 65% A, 35% B; 100–135 min, 85% A, 15% B; and 135–150 min, 85% A, 15% B. Ginsenosides were detected at 203 nm. The sample injection volume was 20 μL, and the temperature of the column was maintained at 35 °C. Both protopanaxadiol (PPD)-type (Rb1, Rb2, Rb3, Rc, Rd, Rg3, Rh2, and CK) and protopanaxatriol (PPT)-type (Re, Rf, Rg1, and Rh1) ginsenosides (ChromaDex, USA) were used as standards. The ginsenoside content was calculated as follows:
2.3. Genotyping using random amplified polymorphic DNA (RAPD) marker analysis 2.3.1. DNA isolation Fresh 6-wk-old adventitious roots (0.1 g) were harvested from selected putative mutant lines and control (non-irradiated) tissues and homogenized using a Tissuelyser II (Qiagen, Germany). Genomic DNA was isolated from root samples using the cetyl trimethyl ammonium 17
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Ginsenoside content (mg·g−1 DW) = ginsenoside concentration (mg·L−1) × sample volume (L) / root DW (g)
CS20 (data not shown). Therefore, the 20 Gy dose was considered as optimal for the selection of mutant lines in ginseng. In this study, four mutant root lines (1G-20-12, 1G-20-16, 1G-20-19, and 1G-20-20) were selected from AR1 culture; these lines showed the highest growth rate at 20 Gy, high branching, and vigorous growth after 40 days of culture on solid MS medium (data not shown).
Total ginsenoside content was calculated as the sum of the 12 individual ginsenosides. Ginsenoside productivity was calculated as follows: Ginsenoside productivity (mg·L−1) = [Total ginsenoside content (mg·g−1 DW) × harvested root DW (g)] / volume of culture medium (L)
3.3. Identification of mutants using RAPD markers To identify DNA polymorphisms induced by gamma irradiation in mutant adventitious root lines, we used RAPD markers. Ten primers were screened in a preliminary analysis, of which four primers showed monomorphic bands. Among the remaining six primers, four primers (A5, A8, A9, and A10) showed highly polymorphic banding patterns, ranging from 300 to 3000 bp (Fig. 4A). To evaluate the genetic variation induced by gamma irradiation, a binary matrix of genetic distances was calculated based on the results of RAPD analysis (Table 2). RAPD data were used to create a dendrogram, which showed two distinct clusters. One of these clusters included two mutant lines (1G-20-20 and 1G-20-19) and the control, while the other cluster comprised 1G-20-12 and 1G-20-16 mutants, indicating higher distinctness from the other root lines (Fig. 4B).
2.6. Statistical analysis The presence or absence of DNA bands on the gel following PCR amplification using random amplified polymorphic DNA (RAPD) primers was scored as 1 or 0, respectively. These scores were entered into a binary matrix and analyzed using NTSYSpc 2.1. Genetic similarities among mutant lines and the control were calculated using Jaccard’s coefficient, and a dendrogram was established using unweighted pair group method with arithmetic mean (UPGMA). Data were analyzed using SPSS version 16.0 (SPSS Inc., USA). Where a significant difference (P < 0.05) was observed for a measured parameter, means were separated using Duncan’s multiple range test at 5% level of significance.
3.4. Growth characteristics of mutant root lines 3. Results The growth characteristics of the four mutant root lines were determined after 40 days of culture in flasks using liquid MS medium (Figs. 4C and 5 ). The selected mutant root lines showed more vigorous growth than the control. However, biomass production showed no significant difference among the mutant lines (Fig. 5A). The length of lateral roots was similar among all mutant root lines and the control, whereas the number of secondary roots was higher in mutant lines than in the control (Fig. 5B, C). In addition, the mutant line 1G-20-19 showed the longest lateral roots. The diameter of lateral roots was higher in the mutant lines 1G-20-19 and 1G-20-20 than in the other mutant root lines and the control (Fig. 5D).
3.1. Effect of 60Co gamma irradiation on the survival of adventitious root and callus in ginseng Owing to long-term maintenance, the survival rate of CS20 was significantly lower than that of the short-term callus (CS1) after treatment with different doses of gamma rays, and CS20 was more sensitive to gamma rays than CS1 (Figs. 1A and 2 ). The survival rate of both short- and long-term callus was the highest at 20 Gy (100% and 75%, respectively). At 30 and 50 Gy, the survival rate of CS1 was maintained at 100% but that of CS20 was reduced to 50% (Fig. 2). At 75 and 100 Gy gamma rays, the survival rate of CS20 was dramatically reduced, whereas that of CS1 declined only slightly (Fig. 2). These data suggest that the survival rate of ginseng callus is unaffected at 20 Gy gamma rays but inhibited at 30 Gy for CS20 and 75 Gy for CS1. The sensitivity of adventitious roots of wild ginseng to gamma rays was evaluated by comparing the lethal dose of gamma rays between long-term and short-term root cultures (Figs. 1B and 3 ). Highly significant differences were recorded among the different doses of gamma rays (Fig. 3). The rate of lethal rate of adventitious roots in both AR1 and AR20 cultures continued to increase with the increase in gamma dosage after irradiation (Fig. 3). Additionally, the AR20 culture was more sensitive to gamma irradiation than AR1 (Fig. 3). The lethal dose at which 50% of the cultures survived (LD50) compared with the control was 52.3 Gy for AR1 and 23.7 Gy for AR20, as shown by the best fit and broken line (Fig. 3). The frequency of genetic mutation and necrosis increased with the increase in the dosage of gamma irradiation (Fig. 3).
3.5. HPLC analysis of ginsenoside content The total ginsenoside content, ginsenoside productivity, and the content of 12 individual ginsenoside compounds (Rb1, Rb2, Rb3, Rc, Rd, Re, Rf, Rg1, Rg2, Rg3, Rh2, and CK) were compared among the four mutant root lines and the control (Table 3, Figs. 6 and 7). The total ginsenoside content was dramatically enhanced in the four mutant root lines compared with the control; the content of PPD- and PPT-type ginsenosides in mutant root lines was 2.3–4.2-fold and 6.2–9.0-fold higher, respectively, than in the control (Table 3). Among the PPD-type ginsenosides, Rg3 in control, 1G-20-12, and 1G-20-20, and Rh2 in 1G20-16, were undetected by HPLC (Fig. 6A). All four mutant lines showed enrichment of Rb2; the level of Rb2 in the mutant lines was 42.2–90.4-fold higher than in the control. Additionally, the ginsenoside Rb1 in 1G-20-16, 1G-20-19, and 1G-20-20 lines was enhanced by 191.9-, 280.8-, and 239.4-fold, respectively, compared with the control (Fig. 6A). Results of HPLC analysis also showed the enrichment of PPTtype ginsenosides in mutant roots compared with the control (Fig. 6B). The ginsenoside Re in mutant roots was enhanced by 79.2–136.3-fold compared with the control; the highest level of Re was identified in the mutant line 1G-20-19. Levels of Rg1 and Rg2 were the highest in mutant lines 1G-20-16 and 1G-20-12, respectively (Fig. 6B). Overall, the ginsenoside production in all four mutant lines was higher than that in the control.
3.2. Formation of adventitious roots from callus and AR cultures and selection of mutant adventitious root lines After gamma irradiation, callus was transferred to adventitious root induction medium for 30 days. The rate of adventitious root formation from both CS1 and CS20 was less than that from AR1 and AR20 cultures, and the roots were shorter, smaller, and slow growing. Each lateral root detached from AR1 and AR20 cultures and irradiated with different doses was considered as one line. The AR1 culture showed more lateral root formation than AR20. A total of 138 lines were isolated from AR1 and AR20 cultures treated with different doses of gamma rays. The frequency of adventitious root formation was highest in AR1 treated with 20 Gy gamma rays compared with AR20, CS1, and
4. Discussion The positive response of callus and adventitious root cultures to low doses of gamma irradiation is consistent that low doses of irradiation 18
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Fig. 1. Effect of different doses of gamma ray in 24 h of short- and long-term callus (A) (CS1 and CS20, respectively) and adventitious root (B) (AR1 and AR20, respectively) Panax ginseng after 4 weeks of treatment (scale bar = 1 cm).
Fig. 3. Determination of LD50 value of gamma rays at different doses for short(AR1) and long-term (AR20) adventitious root cultures of P. ginseng after 30 days of culture. Data represent mean ± SEM of five replications. Dashed lines indicate the LD50 values. AR1, 1-yr-old adventitious root culture; AR20, 20-yrold adventitious root culture.
Fig. 2. Effect of different doses of gamma rays for 24 h on the survival rate of short- (CS1) and long-term callus (CS20) of Panax ginseng at 30 days after treatment. Data represent mean ± standard error of mean (SEM) of five replications. Different letters indicate statistically significant differences (P < 0.05), according to Duncan’s multiple range test. CS1, 1-yr-old callus; CS20, 20yr-old callus.
hormone signaling networks in cells, and uptake of mineral nutrients (Freidman, 1985; Ghiorghita et al., 1985; Antonov et al., 1989; AlOudat, 1990; Kim et al., 2004; Ling et al., 2008). The inhibition of growth caused by higher dosage of gamma rays may be related to the restriction of cell cycle at the G2/M phase and increase in genetic instability (Preuss and Britt, 2003). The survival rate and LD50 values are used to determine the irradiation dosage that is most likely to induce mutations (Mba et al., 2010). While high doses of gamma rays increase the mutation frequency, the lethal rate of mutants also increases; thus, the mutants generated using high doses of gamma rays are less valuable and have lower potential for commercial use (Brunner, 1995). The
have stimulatory effects on the growth of plant tissues (Fowler and MacQueen, 1972; Charbaji and Nabulsi, 1999; Kovalchuk et al., 2007). The lower survival rate and LD50 values of CS20 and AR20 than of CS1 and AR1 indicate that long-term cultures are more radiosensitive than short-term cultures. The enhanced survival rate of ginseng callus and adventitious roots exposed to low doses of gamma irradiation may result from the increased biosynthesis of amino acids (e.g., lysine and phenylalanine), modification of enzyme activities (e.g., catalases and peroxidases), increase in primary metabolite processes, alteration of 19
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Fig. 4. Characterization of mutant adventitious root lines of P. ginseng. (A) QIAxcel gel image of PCR products amplified using random polymorphic amplified DNA (RAPD) primers. (B) Dendrogram of mutant adventitious root lines based on RAPD markers. (C) Morphology of mutant lines after 40 days of culture (scale bar = 1 cm).
gamma irradiation should be identified that induces sufficient mutations while minimizing the negative effects on genome integrity. The physiological features of plant tissues are an important indicator of genetic variation and facilitate the selection of high quality genotypes with enhanced productivity. In the present study, 20 Gy treatment generated mutant adventitious root lines with high biomass, of which four mutant lines, exhibiting a wide range of variability in the number, length, and diameter of lateral roots, were selected (Fig. 5). Physiological characteristics have also been used previously for efficient screening of valuable mutants (de Ronde et al., 2009; Verma et al., 2010). The enhancement of biomass yield and alteration of physiological characteristics in mutant root lines are linked to the level, transport, and signaling of endogenous hormones (George et al., 2008; Atak et al., 2011). RAPD analysis is used for the detection of DNA polymorphisms caused by DNA breakage and adduction, point mutations, and large rearrangements. RAPD analysis has been used for mutant detection in many plant species, including Rosa indica (Chakrabarty and Datta, 2010), Rhododendron (Atak et al., 2011), Jatropha curcas
Table 2 Analysis of genetic similarity among four mutant adventitious root lines of Panax ginseng generated using gamma rays and the untreated control.
Control 1G-20-12 1G-20-16 1G-20-19 1G-20-20 a
Control
1G-20-12
1G-20-16
1G-20-19
1G-20-20
1.00a 0.60 0.69 0.77 0.80
1.00 0.86 0.71 0.63
1.00 0.80 0.71
1.00 0.80
1.00
Binary matrix was analyzed using NTSYSpc 2.1 software.
studies as the cellular level on plants irradiated with gamma rays also showed that high-dose irradiation inhibited the growth remarkably (Wi et al., 2007; Kovács and Keresztes, 2002). The cell membranes in Arabidopsis, particularly thylakoids were heavily swollen and disrupt; the structure of chloroplast was altered; and the concentration of H2O2 was also enhanced in plasma membranes and middle lamellae under highdose gamma radiation (Wi et al., 2007). Therefore, an optimal dose of 20
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Fig. 5. Characterization of growth phenotypes of putative mutant roots of P. ginseng induced by gamma irradiation of adventitious roots after 40 days of culture in Petri dishes. (A) Fresh weight and dry weight; (B) number of lateral roots per explant; (C) lateral root length; (D) root diameter. Data represent mean ± SEM of 5 replications. Different letters indicate statistically significant differences (P < 0.05), according to Duncan’s multiple range test (n = 5).
be caused by alterations in oligonucleotide priming sites, large deletions, and homologous recombination due to mutations (Atienzar and Jha, 2006; Laskar et al., 2018). The loss of bands may be attributed to DNA damage, while the appearance of new bands may be attributed to mutations. The majority of studies on mutation induction have focused on the development of novel lines with higher yield and secondary metabolite accumulation, especially in medicinal plants. In the present study, ginsenoside levels in mutant roots derived from gamma irradiation treatment were higher than in control roots (Table 3). Both PPD- and PPT-type ginsenosides showed higher accumulation in mutant roots, resulting in higher total ginsenoside content and productivity in mutant roots than in the control (Table 3 and Fig. 6). Previously, Kim et al. (2009) showed that mutant ginseng roots accumulate 2.5-fold higher ginsenoside content than wild-type ginseng. The HPLC analysis of mutant lines demonstrated three unknow ginsenoside types. The peak areas of unidentified ginsenoside candidates were higher from 46 to 65% than Rg1, which was the highest content in both mutant and wildtype ginseng (Kim et al., 2009). In this study, the total ginsenoside content of 1G-20-12, 1G-20-16, 1G-20-19, and 1G-20-20 mutant roots
(Dhakshanamoorthy et al., 2011; Dhillon et al., 2014), Acorus calamus (Lee and Han, 2014), Cuminum cyminum (Salarizadeh and Kavousi, 2015), Typhonium flagelliforme (Sianipar and Maarisit, 2015), and Lens culinaris (Laskar et al., 2018). Polymorphisms in genomic DNA are used to evaluate diversity and similarity among samples based on the presence or absence of DNA bands amplified using RAPD primers (Lee and Han, 2014; Ercan, 2015; Salarizadeh and Kavousi, 2015). In the present study, analysis of gamma ray-induced DNA polymorphism using RAPD markers showed differences in the selected adventitious root lines of P. ginseng. The dendrogram results implied that the four selected root lines carried mutations, which distinguished them from the control (Fig. 4). Among the four mutant lines, 1G-20-12 and 1G-20-16 were the most distinct from the control. The number and molecular weight of DNA bands in irradiated mutant root lines of P. ginseng were altered compared with the control. This implies that gamma rays induced severe DNA damage in the majority of the cells in adventitious roots of ginseng. The absence of normal bands might be related to single and double strand DNA breaks, base modifications, DNA-protein crosslinking, point mutations, and chromosomal rearrangements due to gamma irradiation. The presence of extra bands in RAPD profiles may
Table 3 Changes in total ginsenoside content and ginsenoside productivity of mutant Panax ginseng roots by HPLC analysis. Line
Protopanaxadiol (PPD)(mg·g−1 DW)
Protopanaxatriol (PPT)(mg·g−1 DW)
PPD/PPT
Total ginsenoside content (mg·g−1 DW)
Ginsenoside productivity (mg·L−1)
Control 1G-20-12 1G-20-16 1G-20-19 1G-20-20
1.30cz 2.54b 2.05bc 4.58a 3.09b
0.23b 2.09a 1.45a 1.88a 1.48a
5.99a 1.22b 1.41b 2.43b 2.24b
1.53c 4.62b 3.51b 6.46a 4.57b
6.63b 48.61a 51.82a 63.22a 50.49a
z
Different letters within a column indicate significant difference at P < 0.05 according to Duncan’s multiple range test (n = 3). 21
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Fig. 6. Content of individual ginsenosides in mutant adventitious root lines of P. ginseng after 40 days of culture. (A) Protopanaxadiol-type ginsenosides; (B) protopanaxatriol-type ginsenosides. Data represent mean ± SEM of 3 replications. Different letters indicate statistically significant differences (P < 0.05), according to Duncan’s multiple range test (n = 3).
upregulated to gamma rays resulted in the enhancement of ginsenoside content of mutant roots (Kim et al., 2013). Our data showed that the ginsenoside profiles of adventitious roots are affected by the activity of different enzymes in the ginsenoside biosynthesis pathway, the phenotype of adventitious root lines, and culture conditions. Similar results were reported in previous studies (Bonfill et al., 2002; Kim et al., 2009, 2013; Zhang et al., 2011).
were 3.0-, 2.3-, 4.2-, and 3.0-fold higher than that of the control, respectively (Table 3). The content of individual ginsenosides in all four mutant root lines was also higher than that in the control (Fig. 6). The results of Bonfill et al. (2002) imply enhanced biosynthesis of PPD-type ginsenosides due to the presence of auxins, such as IBA. Moreover, it is possible that genes involved in the biosynthesis of PPD-type ginsenosides may be mutated, leading to enhanced levels of these ginsenosides. Additionally, higher content of PPT-type ginsenosides in mutant ginseng roots may be caused by alterations in the activity of principal enzymes involved in their biosynthesis. Kim et al. (2013) showed that the transcription levels of P. ginseng squalene epoxidase and squalene synthase genes were remarkably enhanced by gamma irradiation of ginseng root culture. Dammarenediol synthase, phytosterol synthase, oleanane-type synthase genes were also reported significantly
5. Conclusions Long-term callus and adventitious root cultures of P. ginseng were more sensitive to gamma ray exposure than short-term cultures. Based on DNA fingerprints of adventitious roots, the mutant lines were clustered into two groups; 1G-20-12 and 1G-20-16 were the most diverse
Fig. 7. Chromatogram of 12 standards mixture (A), control (B) and mutant line 1G-20-19 (C) of ginsenoside compounds in Panax ginseng. 22
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from the control. The four mutant lines generated using gamma irradiation were characterized by high biomass yield and efficient production of ginsenosides. The mutant root line 1G-20-19 showed the highest biomass, ginsenoside content, and ginsenoside productivity compared with the control and other mutant lines. Overall, our data suggest that gamma irradiation is a powerful method for increasing the production of valuable secondary metabolites in plants, especially ginsenosides.
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Author contributions Kim-Cuong Le acquired data and wrote the manuscript, and ThanhTam Ho participated in analysis of HPLC. Kee-Yoeup Paek participated in interpretation of data and revision for important intellectual content. So-Young Park conceptualized and designed the study. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. Funding This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through Advanced Production Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (grant number 315013-4). Conflict of interest The authors declare that they have no conflicts of interest. Acknowledgments We thank Dr. Dong-Sub Kim and Mr. Jin-Baek Kim from Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute (KAERI), for technical assistance. References Al-Oudat, M., 1990. Effect of low dose gamma irradiation on onion yield. Ann. Biol 6, 61–67. Antonov, M., Velichov, P., Tsonev, T.S., Angelov, M., 1989. Effect of gamma and laser irradiation on maize seeds and plants. ESNA, XXth Annual Meeting p 44. Atak, Ç, Celik, O., Acik, L., 2011. Genetic analysis of Rhododendron mutants using random amplified polymorphic DNA (RAPD). Pak. J Bot 43, 1173–1182. Atienzar, F.A., Jha, A.N., 2006. The random amplified polymorphic DNA (RAPD) assay and related techniques applied to genotoxicity and carcinogenesis studies: a critical review. Mutat. Res. Mutat. Res. 613, 76–102. Azeez, H., Ibrahim, K., Pop, R., Pamfil, D., Hârţa, M., Bobiș, O., 2017. Changes induced by gamma ray irradiation on biomass production and secondary metabolites accumulation in Hypericum triquetrifolium Turra callus cultures. Ind Crops Prod. 108, 183–189. Baque, M.A., Moh, S.H., Lee, E.J., Zhong, J.J., Paek, K.Y., 2012. Production of biomass and useful compounds from adventitious roots of high-value added medicinal plants using bioreactor. Biotechnol Adv. 30, 1255–1267. Bogdanova, Y., Stoeva, T., Yanev, S., Pandova, B., Molle, E., Burrus, M., Stanilova, M., 2009. Influence of plant origin on propagation capacity and alkaloid biosynthesis during long-term in vitro cultivation of Leucojum aestivum L. In Vitro Cell. Dev. Biol. 45, 458–465. Bonfill, M., Cusidó, R.M., Palazón, J., Piñol, T., Morales, C., 2002. Influence of auxins on organogenesis and ginsenoside production in Panax ginseng calluses. Plant Cell Tissue Organ Cult. 68, 73–78. Brunner, H., 1995. Radiation induced mutations for plant selection. Appl. Radiat. Isot. 46, 589–594. Chakrabarty, D., Datta, S.K., 2010. Application of RAPD markers for characterization of γray-induced rose mutants and assessment of genetic diversity. Plant. Biotechnol Rep. 4, 237–242. Charbaji, T., Nabulsi, I., 1999. Effect of low doses of gamma irradiation on in vitro growth of grapevine. Plant Cell Tissue Organ Cult. 57, 129–132. Choi, S.M., Son, S.H., Yun, S.R., Kwon, O.W., Seon, J.H., Paek, K.Y., 2000. Pilot-scale culture of adventitious roots of ginseng in a bioreactor system. Plant Cell Tissue
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